Production of Dioxygen in the Dark: Dismutases of Oxyanions

DuBois, Jennifer L.; Ojha, Sunil

doi:10.1007/978-3-319-12415-5_3

Jennifer L. DuBois⁶ &
Sunil Ojha⁶

Part of the book series: Metal Ions in Life Sciences ((MILS,volume 15))

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Abstract

O₂-generating reactions are exceedingly rare in biology and difficult to mimic synthetically. Perchlorate-respiring bacteria enzymatically detoxify chlorite (ClO ⁻₂ ), the end product of the perchlorate (ClO ⁻₄ ) respiratory pathway, by rapidly converting it to dioxygen (O₂) and chloride (Cl⁻). This reaction is catalyzed by a heme-containing protein, called chlorite dismutase (Cld), which bears no structural or sequence relationships with known peroxidases or other heme proteins and is part of a large family of proteins with more than one biochemical function. The original assumptions from the 1990s that perchlorate is not a natural product and that perchlorate respiration might be confined to a taxonomically narrow group of species have been called into question, as have the roles of perchlorate respiration and Cld-mediated reactions in the global biogeochemical cycle of chlorine. In this chapter, the chemistry and biochemistry of Cld-mediated O₂ generation, as well as the biological and geochemical context of this extraordinary reaction, are described.

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Keywords

Please cite as: Met. Ions Life Sci. 15 (2015) 45–87

1 Introduction

The S4 state of photosystem II (PSII) – the catalytic species poised to make the O=O bond of O₂ – is one of the most sought-after, and most elusive, chemical structures in the history of biological chemistry [1, 2]. In spite of years of effort and a wide spectrum of approaches, the particulars of how PSII makes this deceptively simple bond are still controversial. At the same time, nature offers few other paradigms for reactions that couple two oxygen atoms. Currently, there is just one other known natural process: chlorite (ClO ⁻₂ ) is converted to chloride (Cl⁻) and dioxygen (O₂) by an enzyme called chlorite dismutase (Cld).

These two systems make for an interesting comparison. PSII is an exceedingly complicated, membrane-associated molecular machine, comprised of around 20 protein subunits (depending on the organism) and a series of cofactors including 35 chlorophyll a molecules, 12 β-carotenes, two pheophytins, two plastoquinones, two hemes, one bicarbonate, 20 accessory lipids, and one non-heme Fe²⁺, all in addition to the famous distorted cubane Mn₄CaO₅ cluster where O₂ evolution takes place [3]. This impressive apparatus harvests energy from light, using it to drive an exquisitely timed series of electron and proton transfers that result in the net splitting of water and emission of O₂ gas.

In sharp contrast to PSII, Cld is a relatively simple, water-soluble heme enzyme that, outside of making an O=O bond with exquisite efficiency, has little else to do [4]. As a consequence, Cld provides a window into one set of electronic and structural requirements for joining a pair of O atoms under biological conditions. Understanding the Cld reaction may therefore yield some analogies for PSII-S4 and perhaps synthetic, structurally still simpler water-splitting catalysts. At the same time, the Cld reaction extends the known repertoire of heme catalysis since, though many heme proteins and model complexes react with chlorite, the issuance of O₂ from any of them is an exceedingly rare outcome. The following chapter describes the structures and chemical mechanisms of the Clds, their diversity, and what is currently known about the geochemical and biological forces that gave rise to this type of catalysis. It concludes with a summary of some of the relevant small molecule and heme protein chemical reactivity with chlorite, asking effectively why they are not “Clds,” and what limits O₂ generation in these cases.

2 Geochemistry of the Oxochlorates

Any thermodynamically well-matched combination of electron source and sink, no matter how exotic, can seemingly be found driving the metabolism of some microbial species. The oxyanions of main group elements such as nitrogen, sulfur, selenium, and arsenic, organic compounds of nitrogen and sulfur, and even solid mineral species (manganese, iron) can all serve as respiratory electron acceptors, provided that an electron donor with a larger reduction potential is available in the same environment. The redox tower diagram illustrates this point, listing several “edibles” (electron sources) and “breathables” (electron sinks) known to be used by various microbial species (Figure 1) [5]. Metabolism of these elements benefits the microbe, and at the same time strongly impacts global geochemical element cycles.

The ability to assume or toggle between different metabolic strategies depends on the presence of the appropriate enzymatic pathways in the microbe’s genome. Some environmental microbes appear to be avid collectors of such pathways, duplicating and transferring them laterally from species to species and providing the raw materials from which new metabolic capabilities can emerge. Evolution, however, depends on the presence of environmental selection pressures to drive it. Microbial perchlorate respiration therefore was a surprise when it was first discovered in the late 1990s [6–11]. At that time, perchlorate was assumed to be almost exclusively a man-made chemical species with few known natural repositories. Wide-scale industrial production of perchlorate as a rocket propellant, constituent of fireworks and explosives, and chemical oxidant moreover only began in the U.S. in 1910 [12], and was increasingly identified as a freshwater pollutant at around the same time that perchlorate-respiring bacteria were discovered [13, 14]. The evolution of microbial perchlorate respiration therefore would have had to occur very quickly in response to man-made perchlorate, or in response to natural sources of perchlorate that had yet to be discovered. Which could it be?

Among other evidence, the identification of perchlorate respiration in diverse microbes from geographically far-flung locales, including places untainted by man-made oxochlorates, now suggests that perchlorate and bacteria that can eliminate it have indeed been with us for a long time. Perchlorate respiration in fact appears to play a pivotal and previously unappreciated role in the global cycling of chlorine. It is also potentially responsible for the dramatically different distribution of perchlorate on the surface of the Earth versus Mars, as described below.

2.1 Oxochlorates as Respiratory Anions

Using perchlorate as a terminal electron sink in respiratory pathways makes great chemical sense. It is highly water-soluble [14], a fact that also colors its ability to distribute in soils and travel in groundwaters [15]. Thermodynamically, it is a powerful electron acceptor. The standard reduction potential for the complete reduction of perchlorate to chloride is on par with the five-electron reduction of nitrate (a more biologically common respiratory anion), or the four-electron reduction of O₂ [14, 16, 17]:

$$ {\mathrm{Cl}\mathrm{O}}_4^{-}+8\kern0.5em {\mathrm{H}}^{+}+8\kern0.5em {\mathrm{e}}^{\hbox{--}}\to {\mathrm{Cl}}^{\hbox{--} }+4\kern0.5em {\mathrm{H}}_2\mathrm{O}\kern1.4em E{}^{\circ}=1.287\kern0.5em \mathrm{V} $$

(1)

$$ {\mathrm{N}\mathrm{O}}_3^{-}+6\kern0.5em {\mathrm{H}}^{+}+5\kern0.5em {\mathrm{e}}^{\hbox{--}}\to \mathrm{\frac{1}{2}}\kern0.5em {\mathrm{N}}_2 + 3\kern0.5em {\mathrm{H}}_2\mathrm{O}\kern0.75em E{}^{\circ}=0.713\kern0.5em \mathrm{V} $$

(2)

$$ {\mathrm{O}}_2+4\kern0.5em {\mathrm{H}}^{+}+4\kern0.5em {\mathrm{e}}^{\hbox{--}}\to 2\kern0.5em {\mathrm{H}}_2\mathrm{O}\kern5.25em E{}^{\circ}=1.229\kern0.5em \mathrm{V} $$

(3)

The successive reductions of chlorate (Cl^5 +) to chlorite (Cl^3 +), chlorite to hypochlorite (Cl^1 +), and hypochlorite to Cl^– are likewise highly thermodynamically favored [14, 18, 19]:

$$ {\mathrm{ClO}}_3^{-}+2\kern0.5em {\mathrm{H}}^{+}+2\kern0.5em {\mathrm{e}}^{\hbox{--}}\to {\mathrm{ClO}}_2^{-}+{\mathrm{H}}_2\mathrm{O}\kern1.7em E{}^{\circ}=1.201\kern0.5em \mathrm{V} $$

(4)

$$ {{\mathrm{ClO}}_2}^{-}+2\kern0.5em {\mathrm{H}}^{+}+2\kern0.5em {\mathrm{e}}^{\hbox{--}}\to {\mathrm{ClO}}^{\hbox{--} }+{\mathrm{H}}_2\mathrm{O}\kern1.3em E{}^{\circ}=1.645\kern0.5em \mathrm{V} $$

(5)

$$ {\mathrm{Cl}\mathrm{O}}^{\hbox{--} }+2\kern0.5em {\mathrm{H}}^{+}+2\kern0.5em {\mathrm{e}}^{\hbox{--}}\to {\mathrm{Cl}}^{\hbox{--} }+{\mathrm{H}}_2\mathrm{O}\kern1.9em E{}^{\circ}=1.49\kern0.5em \mathrm{V} $$

(6)

Biologically, reduction of perchlorate to the product chlorate or chlorate to chlorite occurs with transfer of an oxygen atom, which in each case is protonated to form water. Facilitating proton transfer is consequently likely to be important for each of these reductions. Note that all of the reactants and products are written here as anions, though the standard potentials are reported (pH = 0, versus NHE) and the pK _as of their proton dissociation reactions (HClO_x ⇌ ClO ⁻_x + H⁺) span a very broad range: −10 (ClO ⁻₄ ), −1 (ClO ⁻₃ ), 1.72 (ClO ⁻₂ ), 7.5 (ClO^–), and −8 (Cl^–) [14, 19, 20].

Finally, perchlorate is also a good bet from a kinetic perspective. Like O₂, its reactions with many potential reductants tend to be slow [14]. As a consequence, perchlorate is relatively stable under aqueous/biological conditions. Rather than simply “burning up” in a biological matrix, its thermodynamically favored reduction, like O₂’s, would need to be catalyzed.

2.2 Natural Abundance of Perchlorate on Earth

From a geological perspective, perchlorate seems a decidedly unattractive electron acceptor because it has not accrued to nearly the same extent as O₂ or nitrate, either on land or at sea [21]. Naturally occurring perchlorate at its most abundant can be found comingled with deposits of sodium nitrate in the Atacama Desert of northern Chile (see Figure 2 in Section 2.4). These deposits have been heavily mined as sources for nitrate-based fertilizers. Chilean nitrate is in fact estimated to be the source of ~81 × 10⁶ kg of perchlorate imported to the U.S. (1909–1997) [22–24]. The Atacama itself is a highly unusual, arid, and geologically exotic place. It is believed to have persisted as a nearly rainless climate for 10–15 million years, over which time its nitrate deposits have accumulated. The arid climate and general lack of hydrologic events or large scale biotic (including human) incursions may be responsible for the preservation of the nitrate deposits and for the co-concentration of ions like perchlorate and chromate, which are otherwise not found in large amounts anywhere on earth [25]. These deposits have been shown to have a distinct oxygen isotope pattern, making it possible to distinguish natural and synthetic perchlorate [26].

Less extreme arid and semi-arid North American environments have been surveyed for perchlorate. Many of these have also accumulated the ion in scarcely detectable, very small amounts: for example, on the order of 1–10 μg kg⁻¹ dry soil, where chloride might also be present at 3 or more orders of magnitude greater concentrations [27–30]. Considerably higher (100-fold) concentrations of perchlorate have been found in the very arid soils of the Antarctic [30]. Accumulation of the anion there may have occurred for the same reasons as in the Atacama: that is, because the land has remained extremely dry and unperturbed by either human or microbial activity [31].

The detection of perchlorate in areas where it might have remained undisturbed over long time periods suggests that there are indeed natural mechanisms for perchlorate production. These mechanisms must have been at work for at least several thousand years. Perchlorate has been detected in ground waters deposited thousands to tens of thousands years ago and in glacial ice dating as far back as 2000 years [32–34]. Results from surveys of ground water, ice, and relatively unperturbed deserts have been used to estimate a 0.1–3 × 10⁹ kg “global inventory” of natural perchlorate presently on Earth [22, 28, 33].

Anthropogenic contributions of perchlorate to the environment are relatively recent and far less subtle. The redistribution of natural, Chilean perchlorate stowed away with the nitrate fertilizers has already been mentioned. Industrial perchlorate production from 1951 to 1997 in the U.S. has been estimated as 5 × 10⁹ kg [22]: a figure on the same order as the Earth’s entire estimated inventory of natural perchlorate. Man-made perchlorate has been used in the production of rockets, missiles, fireworks, and the like, where perchlorate’s oxidizing power allows it to be used as a propellant and explosive [12]. Environmental surveys in the early 2000s identified perchlorate as a widespread contaminant in drinking water, crops, and milk, particularly in areas of the southwest United States where perchlorate had been produced or used [16, 21, 35–41]. Microbial perchlorate respiration was documented at around the same time [42, 43], and its origins were then unclear. The identification of perchlorate-respiring bacteria in non-contaminated sites and of perchlorate reservoirs in places predating human activity both suggest that natural perchlorate supplied the initial selection pressure needed for the evolution of perchlorate respiration. This suggestion has gained further support as perchlorate-reducing enzymes have been identified in both taxonomically broad and temporally ancient reaches of the tree of life (see Section 3.1.)

2.3 Atmospheric and Extraterrestrial Origins of Perchlorate

Natural perchlorate may be accruing on the Earth’s surface, but there remains no documented geological or geochemical means of producing it. It would appear as if perchlorate simply falls out of the sky. This suggestion is not so facetious, as the identification of perchlorate in pristine and geographically widespread ground waters, ice cores, rain, and snow indeed suggests atmospheric origins [34, 44]. Three atmospheric sources of perchlorate have been proposed:

First, laboratory experiments with simulated lightning have shown that perchlorate is produced, albeit less efficiently than nitrogen oxides, in oxygenic atmospheres in the presence of Cl^– [45]. Higher voltages and lower humidity, conditions found in high energy storms in dry locales, promote the greatest yields [45]. Second, reactions of aerosolized chlorine and oxygen species, energized by ultraviolet light, have long been known to occur. For example, perchlorate can be formed from reactions between ozone (O₃) and chloride. As a consequence, perchlorate has been proposed to be a stable sink for chloride in the stratosphere, where because of kinetic stability it is expected to resist decomposition by photolytic processes [46, 47]. Perchlorate’s presence in the stratosphere has since been experimentally verified by single particle mass spectrometry on research flights at altitudes of up to 19 km [46]. Such atmospheric reactions would be a highly probable source of continuously deposited, natural, terrestrial perchlorate. Third and finally, in addition to these atmospheric sources, localized deposits of perchlorate have been proposed to have been brought to the Earth’s surface by meteors [28].

2.4 Perchlorate on Mars

In 2008, the Phoenix Lander spacecraft, with the Wet Chemistry Laboratory (WCL) onboard, landed on the surface of Mars (Figure 2) [153]. The WCL analyzed three separate cubic centimeter soil samples for their water-soluble constituents. Quite stunningly, ClO ⁻₄ was found to be their major anionic constituent (0.4–0.6 % by mass) [48].

The hyperarid Martian surface environment is not unlike the Atacama Desert or the dry valleys of the Antarctic, places on earth which have served as Mars surrogates in preparation for missions and places where perchlorate has also accumulated. How Martian perchlorate is generated is not currently known. It could be produced by atmospheric processes similar to those on earth, though the source of the oxygen atoms (O₂, O₃, CO₂, H₂O) is not clear. Mineral-catalyzed photochemical processes, using chloride and water as chemical ingredients, have also been proposed and given some Earth-bound experimental support [49]. Such processes would take advantage of the intense UV light permitted by the thin ozone layer around Mars.

Even if perchlorate production on Mars occurs at a similar or even slower rate than on Earth, the complete lack of hydrological or biological forces for its removal would certainly allow for it to accrue on the Martian surface. Whether this perchlorate could or ever did serve to support Martian microbial life, whether it could serve as a chemical feedstock for O₂ production, and what it tells us about the chemistry of the Martian atmosphere are all open questions for future discussion and perhaps even experimental work.

3 Perchlorate Respiration

3.1 Diversity of Perchlorate-Respiring Microbes

The first dissimilatory (i.e., respiratory) perchlorate-reducing bacteria were isolated in 1996 [42, 43], touching off research both into natural sources of environmental perchlorate (above) and the origins of perchlorate metabolism. Since 1996, ≥50 strains using chlorate or perchlorate as respiratory electron acceptors [collectively, “(per)chlorate”] have been discovered (Figure 3) [50]. The overwhelming majority of these are β-Proteobacteria isolated from freshwater, mesophilic, neutral-pH environments [6, 11, 51, 52].

It has been debated whether these species encompass the real diversity of perchlorate respirers, or whether they merely reflect where the light of investigation has shone most brightly. There is some evidence that the latter phenomenon may be at work. Sampling in a marine environment, for example, yielded the first ε-Proteobacterial dissimilatory perchlorate-respiring bacterium, from the Arcobacter genus [53]. How far this metabolism extends beyond the Proteobacteria, into Gram-positive bacteria or Archaea, for example, is not clear, as the current evidence is incomplete. Moorella perchloratireducens and Sporomusa strain An4 (Gram-positives, phylum Firmicutes) were identified from an underground gas storage facility and shown to have perchlorate-reducing activity [54, 55]. Similarly, strains of Bifidobacteria (Gram-positives, phylum Actinobacteria) have been shown to reduce perchlorate. In each case, though reduction of perchlorate was observed, respiration of the anion has not been verified nor have the genetics of reduction been probed [56]. Because the natural atmospheric events leading to perchlorate production would have existed perhaps deep in prehistory, evidence of perchlorate reduction was sought – and found – in an ancient organism. Archaeoglobus fulgidis is a hyperthermophilic Archaean from extreme subsurface sulfate-reducing environments that resemble conditions on early Earth. This organism appears to possess both genes encoding a perchlorate reductase enzyme (below) and the physiological capacity to use perchlorate as a respiratory anion [57].

3.2 Genetics and Genomics of Perchlorate-Respiring Microbes

Perchlorate-respiring bacteria convert perchlorate to O₂ and Cl⁻ via two enzymes: perchlorate reductase (Pcr) and chlorite dismutase (Cld). Pcr is encoded by a four-gene operon (pcrABCD) that is functionally similar and homologous to the operon that encodes respiratory nitrate reductase (narGHJI) in diverse bacteria. In some cases (examples include Ideonella dechloratans [58] and Pseudomonas chloritidismutans AW-1 [59]), bacteria may respire chlorate but not perchlorate. The responsible enzyme in these organisms is consequently called a chlorate reductase (Clr). It has been experimentally shown that Pcr enzymes can reduce chlorate; however, Clr appears to be unable to reduce perchlorate. These differences in substrate specificity may reflect the far greater kinetic lability of chlorate, which can in fact be reduced by several enzymes including Nar and selenite reductase (Ser), as well as abiotically by Fe(II) or Mn(II) [60, 61]. The ultimate end products of (per)chlorate respiration are water and chlorite. Though the latter anion could in principle be further reduced to provide yet more respiratory energy, it is not reduced. Instead, it is simply detoxified, via the enzyme Cld.

In spite of the similarity of the reactions they catalyze, the Pcr and Clr enzymes are encoded by distinct genes with separate evolutionary origins. The sequences of the PcrA and ClrA subunits, where the oxochlorate anions are reduced, place both enzymes into the dimethyl sulfoxide (DMSO) reductase class (Figure 4). This class can be further subdivided into types I, II, and III, corresponding to three distinct phylogenetic clades [62]. The type I enzymes include periplasmic nitrate reductase (NapA), assimilatory nitrate reductase (NasA), and arsenite oxidase (AsoA). PcrA and ClrA are both type II enzymes, along with selenate reductase (SerA) and the dissimilatory/respiratory nitrate reductase (NarG). However, within the type IIs, the PcrA subunits form their own monophyletic group that is most closely related to the NarGs. The ClrA subunits, by contrast, are more closely related to SerA [50]. Additionally, the four Clr- and Pcr-encoding genes occur in a different order relative to one another (pcrABCD versus clrABDC), where the gene ordering is conserved between Pcr/Nar and Clr/Ser, respectively.

Genomic analyses support the conclusion that perchlorate and chlorate metabolic pathways are genetically and etiologically different, as well as primed for horizontal transfer between species. The first perchlorate-reducing organism to have its genome sequenced was Dechloromonas aromatica strain RCB [63]. Genome sequences for Azospira suillum strain PS [64], the α-Proteobacterium Magnetospirillum bellicus strain VDYT [52], Dechloromonas agitata strain CKB [63], and Dechlorosoma suillum PS [65] have since followed. Similarly, the genomes of five chlorate respiring bacteria (Ideonella dechloratans, Pseudomonas sp. strain PK, Dechloromarinus chlorophilus NSS, Shewanella algae ACDC [66], and Alicycliphilus denitrificans [67, 68]), have been sequenced [66]. Each set of genomes has been compared in order to better understand the evolutionary origins of perchlorate and chlorate metabolism, and the relationships between these two [65]. In perchlorate-respiring bacteria, the Pcr- and Cld-encoding genes are located on a genomic island. In chlorate respirers, the Clr and Cld encoding genes are on a “composite transposon,” a region of the genome flanked by so-called insertion sequences. Insertion sequences are hot spots for bacterial evolution, serving as sites for homologous recombination and plasmid integration. Even among the relatively small available sample set, the transposon varies in its location (genome versus plasmid), copy number, and composition.

The cld gene in all of these organisms is, genomically speaking, something of a rogue. In perchlorate respirers, it appears to assume a number of possible orientations relative to the pcrABCD operon, and its transcription may be under separate regulation [69, 70]. Based on incongruence between protein and species phylogenies, cld genes have been predicted to have undergone horizontal transfer between species on several occasions [71, 72]. A functional chlorite-degrading chlorite dismutase protein was isolated, for example, from Candidatus Nitrospira defluvii: a nitrite-oxidizing bacterium without a (per)chlorate reductase and without known (per)chlorate-respiring capabilities. Genetic evidence suggests that the cld gene in this species was transferred from a taxonomically distant (per)chlorate respirer. The genomic analyses described above suggest that chlorite dismutase originated in the perchlorate respiratory islands, from which it has likely been uprooted and transferred (along with peripheral genetic sequences) to the composite transposons in the chlorate respirers [66]. The movement of a cld gene effectively completes the chlorate respiratory pathway. Such transfers appear to have happened on more than one occasion, again even in the relatively small pool of available genomic sequences.

Some chlorate respirers may have an additional cld gene that is not associated with the chlorate reductase transposon. These clds form a lineage, sometimes called “lineage II”, that is closely related to but nonetheless evolutionarily distinct from the longer, respiration-associated lineage I proteins. Additionally, Proteobacteria with no known oxochlorate respiratory pathways – for either perchlorate or chlorate – may also contain a lineage II Cld. The chemical properties and biological roles of these “Clds” are not clear and are currently under investigation (below).

3.3 (Per)chlorate Reductases

Perchlorate reductases, like nitrate reductases, are molybdenum-dependent oxo-transferase enzymes. Molybdenum in the protein’s α-subunit is coordinated to the thiolates of two pterin cofactors. Each pterin, in turn, is connected to a guanine dinucleotide by a pyrophosphate linkage, and the whole cofactor is known as bis(MGD)Mo [bis(molybdopterin guanine dinucleotide)-molybdenum]. The molybdenum may additionally have a variable number of hydroxide, carboxylate, and cysteine or selenocysteine ligands. In PcrA, the protein-derived ligand is likely a conserved aspartate. Generally speaking, proteins in this family catalyze the transfer of an oxo-atom to or from a substrate. The molybdenum is the holder of two electron equivalents in this transaction, cycling between Mo^IV and Mo^VI. Hence, the complete perchlorate reductase reaction cycle is:

$$ \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{IV}}+{\mathrm{ClO}}_4^{-}\rightleftharpoons \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{VI}}\kern-0.3em =\kern-0.3em \mathrm{O}+{\mathrm{ClO}}_3^{-} $$

(7)

$$ \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{VI}}\kern-0.3em =\kern-0.3em \mathrm{O}+2\kern0.5em {\mathrm{e}}^{\hbox{--} }+2\kern0.5em {\mathrm{H}}^{+}\rightleftharpoons \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{IV}} + {\mathrm{H}}_2\mathrm{O} $$

(8)

$$ \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{IV}} + {\mathrm{ClO}}_3^{-}\rightleftharpoons \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{VI}}\kern-0.3em =\kern-0.3em \mathrm{O}+{\mathrm{ClO}}_2^{-} $$

(9)

$$ \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{VI}}\kern-0.3em =\kern-0.3em \mathrm{O}+2\kern0.5em {\mathrm{e}}^{\hbox{--} }+2\kern0.5em {\mathrm{H}}^{+}\rightleftharpoons \mathrm{bis}\left(\mathrm{M}\mathrm{G}\mathrm{D}\right){\mathrm{Mo}}^{\mathrm{IV}} + {\mathrm{H}}_2\mathrm{O} $$

(10)

In respiratory pathways, reduction reaction occurs at the end of an electron transport chain. Passage of electrons to the substrate is used to generate a proton gradient and, in turn, ATP. Here, perchlorate is initially reduced in a two-electron, water-producing step to chlorate (eqs 7 and 8). A similar Pcr- or Clr-mediated two-electron step reduces chlorate to chlorite (eqs 9 and 10). The electrons in each case come from the electron transport chain embedded in the bacterial inner membrane. These reducing equivalents ultimately originate from a variety of carbon sources that the (per)chlorate-respiring bacteria use as food.

The molybdopterin active site is housed in the α-subunit of Pcr, Clr, or Nar, encoded by PcrA, ClrA or NarG, respectively. Though the subunits are homologs, PcrA or ClrA are considerably smaller (60–104 kDa versus 104–150 kDa) and hence distinct from NarG, making it possible to predict from the sequence (even without computational analysis) that an operon could be involved in oxochlorate metabolism. PcrA- and ClrA-encoding genes moreover begin with a twin-arginine signal peptide, which target the Pcr and Clr enzymes for transport to the periplasmic space in their folded form.

The PcrA, ClrA, and NarG subunits interface with a second, iron-sulfur cluster containing a β-subunit, encoded by pcrB, clrB, or narH. The purpose of the β-subunit is to relay electrons from the respiratory electron transport chain to the α-subunit. Clr and Nar have a further, heme-containing γ-subunit (clrC, narC) that anchors the whole complex to the inner membrane and serves as an electron conduit (Table 1). Pcr, by contrast, appears to be a freely diffusing periplasmic enzyme. Electron equivalents originating from the membrane are predicted to reach the FeS clusters through a soluble, cytochrome b-containing γ-subunit (pcrC) and electron transporting mediators [73].

Table 1 Characterized chlorate and perchlorate reductases.

Full size table

Finally, the δ-subunit of each of these protein complexes (encoded by pcrD/clrD) is a chaperone involved in maturation of the enzyme and possibly in insertion of the bis(MGD)Mo cofactor.

3.4 Chlorite Dismutases and Perchlorate Respiration

All bacteria with a functional Pcr or Clr have a cld gene that encodes a bona fide, functioning chlorite dismutase: that is, a protein that detoxifies chlorite by converting it to Cl⁻ and O₂. In the absence of this reaction or some other means of eliminating chlorite, this end product would accrue to a toxic level. Interestingly, the only known perchlorate-respiring Archaean, A. fulgidis, is able to overcome chlorite toxicity even though it lacks a cld gene. How A. fulgidis degrades chlorite is not known, but abiotic means have been proposed.

The gene encoding the respiration-associated Cld, as described above, is near to the pcrABCD or clrABDC operon but not necessarily in a static location [66]. The properties of these O₂-generating proteins are described in detail in Section 4. Notably, many species with no known (per)chlorate metabolism possess a cld gene. Current evidence suggests that these non-respiratory Clds are not efficient detoxifiers of chlorite and that they serve other cellular roles, as discussed below.

4 Oxygen Generation by Chlorite Dismutases

4.1 Structures

4.1.1 Primary Structures: Diversity and Hallmarks of O₂ Generation

For an organism to respire chlorate/perchlorate, some means of chlorite degradation is necessary because chlorite is toxic. However, the presence of a cld gene does not correlate with either the possession of (per)chlorate reductase encoding genes, or with the ability to reduce oxochlorates. The “lineage I” and “lineage II” Clds were described above as two closely related but distinct groups of cld genes found thus far primarily in Proteobacteria. When a rainbow of microbial genome sequences began to emerge in earnest in the early 2000s, cld genes could be found in a great number of them. In fact, cld-family genes have been identified in highly diverse microbes from at least 9 bacterial and 2 archaeal phyla (Figure 5) [71]. They cluster into at least three additional monophyletic sequence groups beyond lineages I and II.

The respiration-associated Clds come from lineage I, and these proteins all rapidly emit O₂ when presented with chlorite (Figure 6, Table 2). The well-studied Cld from Dechloromonas aromatica (DaCld) survives an impressive 20,000 turnovers with this often-punishing oxidant before its heme irreversibly lyses. By contrast, Clds from diverse bacteria beyond the Proteobacterial phylum – for example, Staphylococcus aureus (Gram-positive pathogen, phylum Firmicutes) and Thermus thermophilus HB8 (Gram-negative hyperthermophile, phylum Deinococcus-Thermus) have no and very little chlorite-degrading capacity, respectively, and their parent organisms do not respire oxochlorates [74, 75]. So, do all Clds from non-respirers fail to produce O₂? Not exactly. Clds from two nitrite-oxidizing, non-(per)chlorate-respiring bacteria were shown to convert chlorite to dioxygen [71, 76], and they illustrate two known types of exceptions. The first of these, the Cld from Candidatus Nitrospira defluvii, is clearly a lineage I Cld that appears to have arrived in the N. defluvii genome via horizontal transfer from a perchlorate respiring organism. The environmental selection pressures promoting its transfer and retention in its new host are not yet understood. The second of these is the Cld from Nitrobacter winogradskyi, which is representative of the lineage II Clds that are found predominantly or perhaps exclusively in Proteobacteria. Though the lineage II clds occur in genomes independently of (per)chlorate reductase genes, the N. winogradskyi Cld and potentially other members of this group (including the Cld from the pathogen Klebsiella pneumoniae) can produce O₂ from chlorite. The number of turnovers sustained before the heme is irreversibly destroyed, however, is sharply limited. Hence, while chlorite catalysis is observable, the catalyst is not robust, suggesting that chlorite decomposition may not be the biological function of the lineage II proteins.

Table 2 Kinetic constants for the steady state reaction of chlorite dismutases.

Full size table

This conclusion is consistent with the lack of a known biological imperative for chlorite detoxification by their parent organisms, although it is possible that these Clds catalyze detoxification of chlorite produced by Nar-mediated chlorate (ClO₃ ⁻) reduction. In short, the available evidence suggests that only the respiration-associated Clds – encoded by genes on perchlorate reductase islands or composite transposons near pcrABCD/clrABDC operons, or recently mobilized from such – efficiently convert chlorite to O₂ and Cl⁻. Cld homologs from outside this relatively small group are biochemically distinct and appear to serve other biological roles.

Sequence alignments including lineage I, II, and the more taxonomically broad Clds show that the family as a whole is quite sequence-diverse (Figure 7, Table 3). Only three amino acids are strictly conserved family-wide. These include the proximal histidine ligand to the heme iron (H171 in DaCld, with residue numbering reflecting the structurally characterized mature form of the protein lacking the SecB tag earmarking the protein for secretion to the periplasm) [4]. They also include a tryptophan near the porphyrin binding site (Trp155) and a proline residue important for maintaining the protein’s fold (Pro148). This sequence diversity, the taxonomic breadth of organisms containing the gene, and the presence of several monophyletic groups of Clds suggests an ancient origin for cld in which divergence of the sequence occurs at pace with speciation events. The exception to this pattern comes, of course, for the heavily traveled cld genes associated with oxochlorate respiration [65, 66, 71, 77].

What sequence features distinguish the O₂-generating Clds from their non-O₂-generating counterparts? There are at least three (Figure 7, Table 3). First, in oxochlorate-respiring bacteria, the Cld-encoding sequence is preceded by a SecB peptide, signaling transport of the protein to the periplasm in its unfolded, heme-free form. Clds from the non-respirers, by contrast, are cytoplasmic. Second, both the longer lineage I Clds from oxochlorate respirers and the shorter lineage II Clds share a conserved arginine residue in the pocket above the heme plane. Proteins from each of these groups have been experimentally confirmed to produce O₂ rapidly, even if the lineage I proteins are considerably more robust (i.e., higher numbers of turnovers). The distal Arg residue is not conserved in the various non-O₂-producing branches of the phylogeny depicted in Figure 5, changing for example to serine (Euryarchaeota), glutamine (Firmicutes), or asparagine (Actinobacteria). Surprisingly, the arginine can be mutagenically substituted with an asparagine in DaCld without completely abrogating activity with chlorite [78]. However, the Cld from Staphylococcus aureus (phylum Firmicutes), which has a distal glutamine residue in the analogous position, has no measurable chlorite-degrading activity. Finally, a second tryptophan near the heme’s periphery (Trp156) and two residues from a triad of hydrophobic amino acids above the heme plane (Leu185 and Phe200) are strictly conserved among all Clds. The tryptophan appears to be critical for maintaining the bound heme as well as the protein’s oligomeric state [79], and hydrophobicity in the distal pocket may be essential for stabilizing reaction intermediates (see Section 4.2.)

Table 3 Conservation of residues of likely functional relevance among the Clds.

Full size table

4.1.2 Heme-Binding Domain

The heme-binding domain of Clds consists of a series of α-helices lining the proximal side of the heme plane and a row of β-sheets over the distal, open coordination position. The iron is ligated by a histidine residue that is strictly conserved throughout the Cld family (Figure 6). In the full length Clds – that is, all Clds except those in lineage II – the protein monomer contains two mutually homologous domains, each about 140 amino acids in length. These resemble one another in secondary structure, with the exception of an added α-helix in the N-terminal domain, and are linked by a pseudo twofold axis of symmetry. The extra helix, the lack of a strictly conserved histidine, and a slightly more compact structure may be responsible for the failure of heme to bind in the N-terminal domain [4, 76, 80–82].

Heme is typically found in predominantly α-helical environments, such as in the globins, cytochrome P450, catalases, and peroxidases. Hence, the Cld heme-binding domain at first seemed somewhat unusual. However, it is now clear that the mixed α-helical/β-sheet domain of Clds is shared by a very large structural superfamily of proteins, many of which derive from microbes and only a small fraction of which associate with heme [75, 83]. Subfamilies range from quite close to Clds in sequence (and therefore ancestry) to relatively distant. At least two subfamilies in addition to the Clds are known to interact with heme. They are catalytically dissimilar to Clds, in spite of their identical heme-binding architecture and mutually conserved residues in the active site. The first subfamily contains the bacterial IsdG-type heme oxygenases that oxidatively cleave heme as a substrate, releasing the iron for cellular use [84]. Unlike Clds, the IsdG-family proteins have just one domain per monomer. Two heme-binding monomers come together to form a dimer that resembles the DaCld bidomain monomer [85]. The heme in IsdI (from Staphylococcus aureus) also assumes a different orientation than in the Clds and is significantly distorted from planarity. Structural distortion of the tetrapyrrole is known to be important for catalysis [86].

The dye-decoloring peroxidases (DyPs) from bacteria and fungi form a second, diverse subfamily. These proteins are evolutionarily quite distant from the Clds, sharing as low as 6 % identity [83]. However, they are structurally very similar, having the same bidomain monomer in which only the C-terminal domain binds heme [87]. In the DyPs, however, the heme is flipped relative to its orientation in the Clds, placing its propionic acid side chains in a very different chemical environment. This again illustrates the remarkable versatility with which this protein domain is known to interact with heme [83]. One of the propionates is able to form a hydrogen bond to the conserved distal arginine. These features and the presence of a distal aspartic acid distinguish the DyPs from the Clds and may be important for their distinct chemistry [87, 88]. DyPs efficiently activate H₂O₂ to form high-valent iron-oxo intermediates which catalyze the one-electron oxidation of substrates. Clds do not, likely because they lack a base for deprotonating H₂O₂ and thereby activating it for binding to Fe(III) [89].

4.1.3 Active Site

Like heme proteins that mediate O₂ transport [90], gas sensing [91–95], and peroxidase catalysis, Clds have a proximal histidine ligand to the heme (Figure 8, left). A glutamic or aspartic acid forms a hydrogen bond to the proximal histidine’s other, unligated nitrogen. Such hydrogen bonding is expected to increase the anionic (histidinate) character of the ligand in peroxidases, making the Fe(III) iron more electron-rich and therefore capable of donating significant electron density toward scission of a peroxide ligand in trans [96, 97]. However, resonance Raman shows that the histidine ligand in Clds, like in the gas-binding proteins, is neutral in character [98]. Also on the proximal side of the heme plane are two tryptophan residues, one strictly conserved throughout the entire Cld family (W155, DaCld numbering) and the other conserved among O₂-producing lineage I Clds and related Clds from lineage II (W156). A third tryptophan (Trp227) is not strictly conserved, but is connected to the proximal histidine through a hydrogen bonding network. Finally, two tyrosine residues (Tyr118, Tyr177) are conserved among the lineage I and II Clds. Tyr118 is connected to a propionate side chain of the DaCld heme by a hydrogen bond. From a structural perspective, either Tyr118 or Trp227 have a direct conduit to the heme and could therefore in principle stabilize radical intermediates, as described below.

The distal side of the heme plane (Figure 8, right) has several unique and likely functionally important features. Notably, there are no distal basic residues (histidine, aspartate, glutamate) in any of the Clds. A distal base is essential for rapid H₂O₂ activation in the well-studied peroxidases [99–101]. As mentioned above, there is instead an arginine directly above the heme plane of all lineage I and II Clds, changing to a serine or glutamine in most other Clds. This residue has no amino acid hydrogen-bonding partner in DaCld or in any available crystal structures of O₂-evolving Clds, and is instead found hydrogen-bonded to the Fe(III) heme ligand (water/hydroxide, nitrite, thiocyanate). Adjacent to the distal arginine are three hydrophobic residues: Leu185, Thr198, Phe200. Although the exact identity of the residues can vary, a hydrophobic triad is strictly conserved at these positions. They form a low, hydrophobic ceiling above the porphyrin plane in what is overall a fairly hydrophobic pocket.

While all chlorite-degrading Clds that have been crystallographically characterized also have a heme bound, a few from non-chlorite-degrading species have been characterized in their heme-free, apo forms (Table 4). These were prepared mainly by structural genomics consortia, and hence the protein expression/purification conditions may not have supplied sufficient heme. However, it has also been observed that the Cld from Staphylococcus aureus, which does not convert chlorite to O₂, binds heme with very low affinity and is actually somewhat difficult to prepare in a heme-bound state [75]. Intrinsically low affinity may be responsible for the failure to capture the Cld structures in the heme-bound form. By the same token, it is notable that these proteins are stable enough to form crystals in the apo/heme-free form, though solution measurements have indicated that the apo-Clds from N. winogradskyi and Candidatus N. defluvii are more prone to denaturation than their holo-counterparts [102, 103].

Table 4 Some available X-ray crystal structures for chlorite dismutases.

Full size table

4.1.4 Tertiary Structures and Oligomerization States

We conclude the discussion of Cld structures by noting the diversity of oligomerization states in which the Cld domain can be found in X-ray crystal structures. All full-length Clds (whether or not they catalyze the chlorite decomposition reaction) form a biochemically unusual homopentameric structure in which the heme-binding domains associate with each other along one face of the pentamer (Figure 9a). Uniquely in DaCld, each monomer-monomer interface has a Ca²⁺ ion ligated by a strictly conserved aspartic acid (Asp192). The pentameric shape of the complex leaves an open, solvent-filled channel in the middle of the protein, the function of which (if any) is not clear. It is also unknown whether the pentameric form is physiologically relevant, although it represents the stable solution state of the pure protein. Full-length Clds in the pentameric state with heme bound and a conserved arginine in the distal pocket are moreover the most efficient catalysts in the conversion of chlorite to O₂ and Cl⁻ [103].

By contrast, the short-length Cld from N. winogradskyi crystallizes as a dimer with a completely different monomer interface (Figure 9b) [76, 82]. This leaves the heme significantly more solvent exposed than in the pentameric structures (Figure 9a). The more open active site may be responsible for the significantly lower stability of the protein’s heme. Though NwCld is capable of producing O₂ from chlorite, catalysis is limited by chlorite-dependent degradation of the heme, which appears to occur at much smaller concentrations of chlorite than in DaCld.

The bacterial heme oxygenases (IsdG, IsdI) and DyPs share the heme binding domain structure of Clds but, as described above, are significantly different in sequence. The bidomain DyPs, unlike Clds, furthermore form head-to-tail dimers, where the C-terminal heme domain of one monomer interacts structurally with the N-terminal heme-free domain of the other. DyP structures may form independent dimers (Figure 9c) or a crystallographic trimer-of-dimers (Figure 9d and e). By contrast, the monodomain IsdG-like proteins form independent dimers in which both monomers bind heme.

This diversity of oligomeric structures demonstrates that the heme-binding domain is structurally capable not only of accommodating heme in multiple orientations, but of interacting in a variety of different ways with partner proteins. Such intrinsic flexibility in their protein-protein interactions could be important for the biological functions or technological applications of diverse Clds.

4.2 Reactivity and Mechanism

4.2.1 Diversity of Reactions Catalyzed by Chlorite Dismutases

Clds catalyze at least three reactions: O–O bond formation, one-electron oxidations (peroxidase chemistry), and heme oxidation.

O–O bond formation from chlorite is the principal reaction catalyzed by full length Clds from (per)chlorate-respiring bacteria. As described above, these “bona fide” Clds are characterized by their pentameric oligomerization state, bidomain monomer structure, and possession of a distal pocket arginine in the heme-binding domain. They constitute a relatively small minority of all the sequenced Clds from taxonomically diverse sources. The small dimeric Clds have nearly the same active site structure as their pentameric homologs, including the distal arginine and the surrounding hydrophobic residues. These Clds also catalyze rapid O–O joining in spite of the lack of a known biological imperative by their host organisms for eliminating chlorite. However, the limited number of turnovers their hemes can sustain suggests that chlorite may not be their natural substrate [76, 104].

Whether short or long, the O₂-producing proteins also appear to be capable of catalyzing peroxidase-type one-electron oxidation reactions when either H₂O₂ or a peracid is the oxidant [89, 104]. A complete peroxidase reaction cycle has been documented for DaCld, as described below. Catalysis of this reaction is very slow and the number of achievable turnovers per heme limited, consistent with the assumption that peroxidase chemistry is not the biological function of these Clds. Studies of the peroxidase reaction nonetheless have shed light on the basic chemical and biological capabilities of Clds and are therefore of interest [89].

Finally, Clds, like many heme-dependent oxidation catalysts, facilitate the oxidant-dependent degradation of their own hemes. The redox-stability of the heme in Clds is extraordinarily variable. DaCld sustains ~20,000 turnovers with chlorite, per heme [105]. The dimeric Cld from Klebsiella pneumoniae sustains only ~2–3,000 turnovers under similar conditions [104]. The heme in the Cld from Staphylococcus aureus is completely destroyed by less than five equivalents of chlorite, without measurable production of O₂ [75]. Whether heme destruction serves a biological function in any of these proteins is not clear. However, it does seem to be the principal catalytic pathway followed by the S. aureus Cld (and likely by related pentameric Clds from non-oxochlorate-respiring bacteria) in the presence of a series of oxidants (chlorite, H₂O₂, peracids).

4.2.2 Possible Pathways for O–O Formation

There are a number of possible pathways for O₂ production from chlorite. Early efforts with DaCld truncated the list by demonstrating that (i) O₂ is produced stoichiometrically with ClO ⁻₂ degraded [105]; (ii) all of the emitted O₂ derives from chlorite, even in the presence of ¹⁸O–labeled water [106]. These results strongly favored mechanisms where the two oxygen atoms of O₂ derive from the same molecule of ClO ⁻₂ . Alternative mechanisms where chlorite acts as an oxygen atom or one-electron donor to the Fe(III) heme produce highly protein-reactive leaving groups (ClO⁻, ClO₂) [107, 108] that would have to re-react quantitatively at the heme site to maintain the observed 1ClO ⁻₂ :1O₂ stoichiometry. They would moreover have to do so without undergoing exchange of their oxygen atoms with ¹⁸OH₂. Hence, the four possible mechanisms by which a single molecule of chlorite can be effectively rearranged to generate Cl⁻ and O₂, depicted in Figure 10, are preferred.

In pathway (1), a single molecule of chlorite binds Fe(III) in a bidentate, η² mode, and formation of O₂ occurs in a concerted fashion without any intermediate transfer of electrons or atoms to the iron. In pathway (2), electron transfer from chlorite to the Fe(III) heme, depicted here as an inner-sphere process, yields the Fe(II) porphyrin and chlorine dioxide (ClO₂). This reaction (eqn. 11) is thermodynamically favorable [109] and known to be catalyzed by certain synthetic metalloporphyrins (described below):

$$ {\mathrm{ClO}}_2^{-}\to {\mathrm{ClO}}_2 + {\mathrm{e}}^{\hbox{--}}\kern3em E{}^{\circ}=\hbox{--} 0.934\kern0.5em \mathrm{V} $$

(11)

Oxygen atom transfer from chlorine dioxide to the Fe(II) iron would yield a ferryl (Fe^IV=O) heme. This could recombine with the released chlorine oxide to make a formally Fe(III)-OOCl⁻ species, spontaneously breaking down to yield Cl⁻ and O₂. The Fe(II) Cld may also react with chlorite to give O₂, which is also depicted in Figure 10.

Pathways (3) and (4) both begin with the formation of a Fe(III) heme/chlorite complex. This complex is analogous to the Fe(III)-peroxy species (Fe(III)-OOH) formed as the initial intermediate in peroxidase reactions, known as Compound 0 [110]. The distal histidine of classical peroxidases removes the proton from H₂O₂ (pK _a = 12) to yield the peroxy anion, which binds avidly to Fe(III). Because the hypochlorous acid/chlorite pK _a is quite low (1.72), base catalysis would not be needed to form the analogous Fe(III)-chlorito complex.

Donation of two electrons from the Fe(III) heme leads to heterolytic scission of the coordinated O–Cl bond (pathway 3) and formation of a formally Fe(V)=O intermediate. This intermediate, observed pervasively in biological heme chemistry, is known as Compound I. Electronically, it is described as a ferryl (Fe(IV)=O) that is exchange-coupled to a radical on the non-innocent porphyrin ligand. Because loss of an electron occurs without concomitant loss of a proton, the resulting porphyrin radical is a cation (porphyrin^• +). Unlike the parent tetrapyrrole, porphyrin^• + is not aromatic. Hence, the porphyrin’s visible Soret absorbance band is significantly diminished in intensity in porphyrin^•+, providing a simple indicator of the possible presence of Compound I. The oxygen atom in Compound I is capable of removing electrons or an electron/proton pair (hydrogen atom) from a substrate, thereby becoming reduced to the ferryl-porphyrin intermediate (Fe(IV)=O or Fe(III)-OH), also known as Compound II [111]. A good nucleophile may also attack the electron-deficient oxygen of Compound I. The oxygen atom of hypochlorite, particularly in its anionic/deprotonated form, is nucleophilic and could attack Compound I to yield the same Fe(III)-peroxychlorite intermediate proposed in pathway (1).

Alternatively, the Fe(III)-chlorite complex could react in a one-electron fashion to give the products of heterolytic O–Cl bond cleavage: Fe(IV)=O and the ClO^• (chlorine monoxide) radical. These two would need to re-react, once again to form a Fe(III) species with an O–O(Cl) bond (pathway 4). Chlorine monoxide is presumably a highly reactive species, perhaps not dissimilar to the hydroxyl radical. It has been described in atmospheric chemistry models but is not a known biological molecule.

There is no precedent for pathway (1) using either synthetic or biological catalysts. In fact, there are very few reported crystal structures for metals bound to chlorite, and none in either the through chloride or bidentate O,O-η² modes. While not a strict necessity, either of these binding modes and particularly the latter would seem to be the most logical for catalyzing the concerted formation of O₂ and Cl⁻. By contrast, there are several direct precedents for parts of both mechanisms (2) and (3). The two long-proposed mechanisms for O–O joining in PSII and in synthetic water-splitting catalysts, for example, involve two oxygen atoms poised to react via either nucleophilic or radical processes [1, 2, 112, 113]. Mechanisms that are chemically analogous to pathways (3) and (4) have also been proposed for Fe(III) porphyrin-mediated isomerization of peroxynitrite (ONOO⁻) to nitrate [114–117]. An Fe(III)-coordinated ONOO⁻ anion could undergo heterolytic O–N bond cleavage to yield Compound I and the anionic leaving group (nitrite). Nitrite would then act as the nucleophile, attacking Compound I to form nitrate. Alternatively, homolytic O–N bond cleavage would yield Compound II and the neutral, radical leaving group (NO₂). Radical joining of NO₂ and the ferryl oxygen then lead to the Fe(III) porphyrin and NO ⁻₃ .

4.2.3 Catalytic Efficiency

Clds that produce O₂ tend to do so very efficiently. Values for k _cat/K _M for several Clds are listed in Table 2, and many are near the diffusion limit. The rapidity with which O₂ is produced has made Clds useful generators of O₂ for stopped flow studies, where oxygen gas is produced within a few milliseconds in an initial mixing experiment between Cld in one syringe and chlorite in another [118]. The oxygenated solution is mixed with a third, concentrated solution of the reduced/anaerobic enzyme species of interest, and the subsequent reaction is monitored. Dioxygen is produced in this way at concentrations of up to 9 mM (25 °C, neutral pH) without observed visible light scattering in a stopped flow reaction chamber, which would be indicative of bubbling. It has been postulated that the solution remains transiently supersaturated over the very short time scale of the initial, O₂-producing mix, although the process of bubble growth has not been explicitly monitored over time.

Having the means to produce very large concentrations of O₂ with rapidity has allowed an important barrier to be surmounted. Namely, many intermediates in reactions between reduced biomolecules and O₂ cannot be observed because they decay more rapidly than they form. However, the second-order rate of their production can be enhanced by increasing [O₂]. Using DaCld in this way, that is, as a reagent, has indeed yielded impressive results in cases previously limited by the rapid decay of the intermediate [118]. Other biotechnological applications of the reaction are currently under development.

4.2.4 Reaction Intermediates

While efficiency is critical for a detoxification enzyme and for the experiments described above, it can be the enemy of mechanistic studies. It has thus far not been possible to detect intermediates in the Cld/ClO ⁻₂ reaction (e.g., Compounds 0, I, II) via stopped flow spectrophotometry. Hence, the Cld mechanism has not been monitored directly. By the same token, addition of a variety of colorimetric reagents in the presence of DaCld during turnover with chlorite – small peroxidase substrates such as guaiacol or ascorbate, or the chlorination substrate monochlorodimedone (MCD) – has not resulted in observable side reactivity [105, 119]. This suggests that potential intermediates such as ClO₂ (ε_{359 nm} = 1.23 mM⁻¹ cm⁻¹) [120], ClO⁻, or possibly ClO^•, any of which would react with MCD, either are not generated or cannot be accessed by MCD. The oxidation of molecules like guaiacol or ascorbate by potential intermediates Compound I or II is likewise not observed.

However, DaCld can oxidize the peroxidase substrates guaiacol, ascorbate, and 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid if either peroxide or peracetic acid is the oxidant. Moreover, the transient reactions of DaCld with these oxidants have been examined [89]. These experiments showed that oxygen atom transfer from the peracid donor is very rapid and leads to the formation of Compound I, even in the presence of just one equivalent of oxidant. Hence, while it remains uncertain whether Compound I is an intermediate on the regular reaction pathway with chlorite, it is clear that the enzyme readily forms this species in the presence of another anionic oxygen atom donor that is thermodynamically predisposed toward oxygen atom transfer. These two donors are not identical, however. Unlike chlorite, for which the leaving group is relatively basic and therefore nucleophilic (hypochlorite, pK _a = 7.5), peracetic acid leaves behind a much less nucleophilic acetate ion (pK _a = 4.5). The reaction between the Fe(III) heme and chlorite would be expected to be catalyzed by a proton donor or positive charge that could stabilize the leaving group. The distal arginine has been proposed to fulfill this role, stabilizing hypochlorite in its anionic form and priming it for re-reaction with the oxygen atom of a hypothetical Compound I.

Interestingly, the actual Compound I formed in the reaction between peracetic acid and Fe(III) DaCld reacts avidly with peroxidase substrates [89]. Under stopped flow conditions, a full peroxidase catalytic cycle is observed, with one equivalent of a one-electron donor (ascorbate) forming Compound II and the second returning the intermediate to the Fe(III) form:

$$ \mathrm{Fe}\left(\mathrm{III}\right)\left(\mathrm{porphyrin}\right)+{\mathrm{CH}}_3\left(\mathrm{C}\kern-0.2em =\kern-0.2em \mathrm{O}\right){\mathrm{COO}}^{\hbox{--}}\kern-0.3em \to \mathrm{Fe}\left(\mathrm{IV}\right)\kern-0.2em =\kern-0.2em \mathrm{O}\left({\mathrm{porphyrin}}^{\bullet +}\right)\kern13em +{\mathrm{CH}}_3{\mathrm{COO}}^{\hbox{--} } $$

(12)

$$ \mathrm{Fe}\left(\mathrm{IV}\right)\kern-0.3em =\kern-0.3em \mathrm{O}\left({\mathrm{porphyrin}}^{\bullet +}\right)+\mathrm{ascorbate}\to \mathrm{Fe}\left(\mathrm{IV}\right)\kern-0.3em \hbox{--} \mathrm{O}\mathrm{H}\left(\mathrm{porphyrin}\right)\kern10.5em +{\mathrm{ascorbate}}^{\bullet } $$

(13)

$$ \mathrm{Fe}\left(\mathrm{IV}\right)\kern-0.3em \hbox{--} \kern-0.3em \mathrm{OH}\left(\mathrm{porphyrin}\right)+\mathrm{ascorbate}\to \mathrm{Fe}\left(\mathrm{III}\right)\left(\mathrm{porphyrin}\right) +{\mathrm{ascorbate}}^{\bullet }+{\mathrm{H}}_2\mathrm{O} $$

(14)

These results demonstrate that DaCld’s Compound I can access peroxidase-like substrates, which in real peroxidase enzymes are known to bind in different locations above and adjacent to the heme plane [121].

By contrast, H₂O₂ reacts much more slowly with Fe(III) DaCld than peracetic acid and, rather than producing Compound I, the only observed spectroscopic changes following the reaction correspond to heme oxidation. This observation is consistent with the Clds’ lack of a distal pocket base. A distal histidine is absolutely required for rapid Compound I formation in the well-studied plant peroxidases [122–125]. When this residue is mutated to an aliphatic amino acid, the rate of Compound I formation slows by five orders of magnitude. In short, DaCld can be a very effective peroxidase, but only in the presence of an acidic oxygen atom donor to the Fe(III) heme, for which a distal base is not required.

The DaCld Compound I intermediate is not long-lived. It spontaneously converts to a ferryl without a coupled porphyrin cation radical. This conversion becomes more rapid at higher pH. The relative instability of Compound I is consistent with the generation of O₂ from a single molecule of chlorite. Namely, if Compound I is an intermediate on the O₂ production pathway, it may be too short-lived to react with oxygen-atom donors that are not already in the vicinity of the ferryl. Indeed, a Compound I species prepared in the stopped flow did not react with exogenously supplied ClO⁻/HClO to form O₂, as the reaction appears to be kinetically out-competed by decay of the Compound I intermediate [89]. Though the pathway has not been studied, Compound I’s breakdown has been presumed to occur via the conserved tyrosines that ring (and in one case form hydrogen bonds with) the heme [79].

Finally, although oxidation of guaiacol/ascorbate or chlorination of MCD is not observed during DaCld/chlorite turnover, the peroxidase substrates increase (~10-fold) the number of molecules of chlorite that DaCld can decompose before it irreversibly inactivates. The high valent intermediates are returned to the Fe(III) state and hence to activity with chlorite by these substrates. A similar type of “rescue” is likewise observed in other heme proteins [126–128]. This suggests that Compounds I, II, or both are intermediates on the pathway to whatever limits the catalytic lifetime of Clds (see below.) By contrast, MCD has no such rescuing effect, suggesting either that diffusible O/Cl species are not produced or are not appreciably responsible for substrate-dependent inactivation of DaCld.

4.2.5 Structure-Activity Relationships: Highlights

The active site of the typical bona fide, chlorite-degrading Cld was discussed above in terms of what it is; it is also worthwhile to consider what it is not. Unlike peroxidases, which characteristically have a histidine-arginine pair in their distal pockets, Clds possess an arginine as the sole conserved, polar residue. The distal histidine is critical for peroxidase function as described above, acting as a base to deprotonate H₂O₂ and then as an acid toward the resulting Fe(III)-OOH (Compound 0) intermediate to generate Compound I and water. Such proton motions are critical for the rapid formation of both Compound 0 and Compound I in other heme proteins that activate O₂ or H₂O₂. Fe(III) Clds lack both a distal basic residue and a recognizable proton conduit to the heme iron. Perhaps not surprisingly, Clds bind anionic CN⁻ and CH₃(C=O)COO⁻ (surrogates for OOH⁻) much faster than protonated HCN or H₂O₂ [104]. DaCld also rapidly forms Compound I following reaction with peracetic acid but not H₂O₂ [89]. In short, Clds lack the necessary machinery for efficiently binding and activating peroxide, and this colors what they can and cannot efficiently do with biological oxidants.

By contrast, Clds readily bind anions, including the substrate chlorite. Here, the distal arginine plays a key role. In multiple crystal structures, it forms a hydrogen bond to the axial ligand bound to the Fe(III) iron, from which it is separated by ~3 Å [4, 76, 80, 82]. Notably, the arginine has no identifiable hydrogen bonding partners among either the active site amino acids or the heme propionates, though an Arg-propionate interaction is present in some DyPs [83]. DaCld, in fact, does not crystalize in the absence of a heme ligand, possibly in part because the motion of this residue would otherwise remain unrestrained. Resonance Raman experiments support a model where the arginine side chain interacts with axially-bound distal ligands and assumes different orientations (“in” or “out,” relative to the heme plane) as a function of pH [78, 98]. In solution, substitution of the arginine with neutral glutamine by mutagenesis results in a strongly diminished k _cat/K _M(chlorite) and in a loss of affinity for anions [78]. This indicates that arginine is indeed critical for stabilizing the Michaelis complex, presumed to be a Compound 0-like Fe(III)-chlorito species [78].

The distal arginine is also sufficient for promoting rapid, heterolytic cleavage of the peracid O–O bond and stabilizing the resulting Compound I intermediate [89]. Similar functions have been attributed to the distal arginine in classical peroxidases, though in conjunction with an anionic proximal ligand to the heme iron. In peroxidases, anionic or histidinate character in this ligand is understood to add electron density to the Fe(III) and to consequently support heterolytic cleavage of a bond coordinated in trans. As described by the classic “push-pull” model of peroxidase catalysis, the electron-rich proximal ligand supplies a “push” toward cleaving a coordinated O–O bond, while the distal arginine supplies a concurrent “pull” [96, 124, 129]. The imidazole ring of the histidine gains anionic character via hydrogen bonding to an aspartate or glutamate. Though DaCld has such a His-Glu hydrogen bond, resonance Raman showed the proximal ligand to be neutral in character [98]. O–O bond cleavage is relatively rapid in DaCld in spite of the nature of the protein-derived ligand on the proximal side of the heme iron, suggesting that the distal pocket’s “pull” alone is enough. The fact that DaCld readily cleaves the O–O bond of coordinated peracetate ion also suggests that, in principle, it should be able to heterolytically cleave the O–Cl bond of a bound chlorite with similar avidity.

The mobility of the arginine could be important for an additional, important function: stabilizing the hypochlorite/hypochlorous acid (aqueous pK _a = 7.5) formed when the O–Cl bond of chlorite is broken. Nucleophilic attack of the leaving group on Compound I should be significantly more avid for the deprotonated hypochlorite than for hypochlorous acid, as described above. The positive charge of the arginine could locally lower the pK _a for the anion/acid pair, stabilizing the hypochlorite. It could also steer the hypochlorite into an optimal position for nucleophilic attack on Compound I.

Finally, the distal arginine could not manage to be such a key player were it not for a supporting cast of conserved, hydrophobic residues surrounding it. These residues constitute a sterically confined, chemically unreactive low ceiling over the heme plane [4]. Such an enclosed environment seems ideal for enforcing the recombination of a hypochlorite leaving group with Compound I.

4.2.6 Heme and Protein Stability in Diverse Chlorite Dismutase Family Proteins

The pentameric O₂-evolving DaCld catalyzes ~20,000 turnovers (per heme) before irreversibly inactivating. The same number of chlorite equivalents completely eliminates the heme chromophore [105]. This suggests that oxidative destruction of the heme is responsible for the observed irreversible inactivation. Indeed, heme scission is a common side effect of oxidative catalysis: playing with matches will eventually cause even a robustly designed protein to get burned.

The mechanism of heme decomposition has not been studied. It has been observed, however, that far fewer turnovers are sustained by structurally distinct Clds [76]. The dimeric Cld from Klebsiella pneumoniae, for example, contains a distal arginine and catalyzes the chlorite to O₂ conversion with steady state parameters similar to those measured for DaCld. However, the lifetime of the enzyme is clearly shorter; the measured turnover number is only 2,000–3,000 per heme [104]. The pentameric Cld from Staphylococcus aureus has a glutamine in place of the distal arginine. Its heme disappears following incubation with fewer than five equivalents of chlorite, and no O₂ evolution from this protein is observed [75]. The structural features leading to the great disparity in heme stability across the Cld family are not understood.

5 Synthetic and Biochemical Models

5.1 Chlorite as Reagent with Related Synthetic Metalloporphyrins

Quite apart from Clds and O₂ generation, NaOCl and NaClO₂ have an illustrious history as reagents in metalloporphyrin chemistry. Like iodosylbenzene, organic peracids (RCO₃H), and KHSO₅, the oxochlorates have been used as so-called “shunt” reagents: oxygen atom donors that, unlike O₂, do not require the input of a pair of activating electrons from the catalyst. Such shunt reagents have been used extensively in studies of cytochrome P450 monooxygenases and their synthetic mimics [130–132]. They have also been used in studies of peroxidases and heme oxygenases, where the oxygen-atom transfers to a Fe(III) heme center to yield Compound I. The same intermediate can equivalently be reached by the addition of O₂, three electrons, and one proton to a Fe(II) heme (P450), or peroxide minus one proton to the Fe(III) heme (peroxidases), with water as the leaving group.

Motivated by the goals of understanding and possibly mimicking P450 chemistry, Collman et al. were the first to describe O₂ evolution from synthetic Mn(III)porphyrin catalysts and ClO ⁻₂ in non-aqueous media [133]. O₂ production occurred as a side reaction to a more P450-like process: the oxidation of unactivated alkanes (e.g., cyclohexane), which were converted in a two-electron process to alcohols and in a four-electron process to ketones. Notably, the turnover numbers using chlorite were >40-fold higher than if other oxygen-atom donors were used, and the ketone/alcohol ratio was at least double. These results suggested that chlorite was acting as something other than a simple oxygen-atom donor to the manganese. Further investigation then showed that radical trapping agents inhibited turnover, indicating that the Mn(III)/chlorite system acted as a radical initiator.

A two-pathway scheme was proposed to explain the observed oxidations of cyclohexane to cyclohexanol and cyclohexanone (Figure 11) [132]. In the first pathway, a Mn(III)/chlorite-derived species was proposed to generate a cyclohexyl radical by hydrogen-atom (H^•) abstraction. The organic radical then combined with O₂ to form a peroxyl radical adduct. The reaction of two such C₆H₁₁-OO^• species would then lead to the formation of cyclohexanol, cyclohexanone, and, notably, O₂. A radical process helped explain the observation of ketone products, which were shown not to arise from the oxidation of alcohols formed initially as precursors. In the second proposed pathway, oxygen atom transfer from chlorite to the manganese(III)porphyrin catalyst (via heterolytic Cl–O bond cleavage) was proposed to yield a formal Mn(V)=O species and ClO⁻ as the leaving group. The high valent Mn(V)=O could then react via a more conventional, P450-like rebound mechanism to form the hydroxylation product:

$$ \mathrm{M}\mathrm{n}\left(\mathrm{V}\right)\kern-0.3em =\kern-0.3em \mathrm{O}+{\mathrm{C}}_6{\mathrm{H}}_6\ \to \mathrm{M}\mathrm{n}\left(\mathrm{I}\mathrm{V}\right)\kern-0.3em -\kern-0.3em \mathrm{O}\mathrm{H}+{\mathrm{C}}_6{\mathrm{H}}_5^{\bullet}\to {\mathrm{C}}_6{\mathrm{H}}_5\kern-0.3em -\kern-0.3em \mathrm{O}\mathrm{H}+\mathrm{M}\mathrm{n}\left(\mathrm{I}\mathrm{I}\mathrm{I}\right) $$

(15)

In addition to the two alkane-oxidizing pathways, simple, metal-catalyzed decomposition of chlorite by two routes was also observed. In the first, Cl⁻ and O₂ were produced. Though a mechanism was not explicitly offered, it is possible that Mn(V)=O and Cl⁻ combined in the same manner proposed for the enzymatic/Cld reaction. The second pathway led to conversion of chlorite to chlorate and chloride in a 3:2:1 ratio:

$$ 3\kern0.5em {\mathrm{Cl}\mathrm{O}}_2^{-}\to 2\kern0.5em {\mathrm{Cl}\mathrm{O}}_3^{-}+{\mathrm{Cl}}^{\hbox{--} } $$

(16)

Because the manganese catalysts are inert toward chlorate, it persisted here as a dead-end product.

The laboratory of Abu-Omar subsequently investigated O₂ generation from metalloporphyrin/chlorite systems as the target reaction rather than as a side process [134–136]. Water-soluble iron porphyrin complexes with different aryl groups at the four meso positions were studied (Figure 12) as models for Clds. The p-sulfonatophenyl- and N,N,N-trimethylanilinium-substituted Fe(III) porphyrins led primarily to the same kind of chlorite-to-chlorate disproportionation observed by Collman et al. (eqn. 16) [133]. This mechanism of the reaction was studied in some detail, and shown to occur via the initial formation of an Fe(IV)=O(porphyrin^•+) (Compound I) intermediate. Compound I was proposed to comproportionate with an aqua-Fe(III)porphyrin, resulting in a pair of Fe(IV)–OH(porphyrin) (Compound II) species. Compound II would then transfer its oxygen atom to chlorite, generating chlorate and the two-electron reduction product of the catalyst, Fe(II)porphyrin. The Fe(II)porphyrin thus formed can react with both chlorite and hypochlorite, heterolytically cleaving their Cl–O bonds and generating further reactive Fe(IV)–OH(porphyrin).

Though the first two iron porphyrins did not produce O₂ from chlorite, the fluorinated analog of one of them, [Fe(III)(TF₄TMAP)](OTf)₅, did in 18 % yield [OTf = triflate, TF₄TMAP = 5,10,15,20-tetrakis(tetrafluro-N,N,N-trimethylanilinium)porphyrinato]. The remaining chlorite followed the more customary pathway of disproportionation to chlorate and chloride. Production of O₂ was proposed to begin with oxygen atom transfer from chlorite to the Fe(III)porphyrin, forming Compound I. The same step was invoked as in the initiating step for chlorate generation. However, circa 1/5 of the time, the Compound I intermediate reacts with ClO⁻ instead of another Fe(III)porphyrin, generating O₂ and Cl⁻. Notably, enclosure of the ClO⁻ leaving group and Fe(IV)=O(porphyrin^+•) species, as well as site-isolation of the reactive metal, would both in principle improve the O₂ yield from the otherwise catalytically competent Fe(III)porphyrin. Isolation of the reactive intermediates is an obvious role served by the Cld protein environment.

In addition to the disporoportionation and O₂ generation reactions described above, some manganese(III)porphyrins catalyze yet a further transformation of chlorite: its one electron oxidation to the chlorine dioxide (ClO₂) radical (Figure 13) [137, 138]. This reaction is preferentially catalyzed by the water-soluble Mn(III) analogs of [Fe(III)(TF₄TMAP)]⁵⁺ and by tetrakis-5,10,15,20-(N,N-dimethylimidazolium) porphyrinatomanganese(III), [Mn(TDMImP)]⁵⁺. In each case, the reaction was proposed to begin with oxygen atom transfer to the Mn(III), generating the Mn(V)=O product of heterolytic Cl–O bond cleavage in the [Mn(TDMImP)]⁵⁺ system and a mixture of the heterolytic and homolytic (Mn(IV)=O) products in the presence of [Mn(TF₄TMAP)]⁵⁺. The ClO⁻ leaving group was able to re-enter the catalytic cycle as an oxygen-atom donor to Mn(III)porphyrin. The ClO^• (product of homolytic Cl–O cleavage) was proposed to react in an uncatalyzed fashion with additional molecules of ClO ⁻₂ . The high-valent metal-oxo species produced in either case served as one-electron oxidants toward chlorite [109], generating neutral chlorine dioxide gas (eqn. 11).

This reaction is interesting in itself from a technological point of view, because ClO₂ is a reagent used in water purification that must be produced near its point of use because it cannot be easily compressed and stored. It also provides a potential mechanistic contrast with Clds and the porphyrin complexes which generate Compound I but appear to use it to transfer an oxygen atom to hypochlorite (a two-electron process) rather than for the one-electron oxidation of chlorite. Presumably, recombination between the Compound I and hypochlorite generated in the confined space above the Cld heme kinetically out-competes the reaction between Compound I and another molecule of chlorite. This is a hypothesis that awaits further experimental investigation.

5.2 Reactions of Chlorite with Horseradish Peroxidase: Implications for Chlorite Dismutases

The use of chlorite as both an oxidant (oxygen-atom donor) and one-electron reductant by the Mn(III)porphyrins mirrors the results of a careful, systematic study of the reactions of horseradish peroxidase (HRP) and chlorite [120]. The complexity of this reaction had been previously noted [139, 140]. In strong contrast to Clds, Fe(III) HRP reacted with both chlorite and hypochlorite as oxygen-atom donors (generating Compound I), followed by two molecules of chlorite serving as one-electron donors to return the intermediate to the Fe(III) state (i.e., a complete peroxidase-like catalytic cycle, eqs 12–14). The chlorine dioxide products were also capable of donating an oxygen atom to the Fe(III) HRP, yielding Compound I and chlorine oxide (Figure 14). The ultimate fate of the highly-reactive ClO^• radical was not clear.

In spite of having a relatively well-enclosed active site that could in principle contain and steer ClO⁻/ClO^• leaving groups, neither appeared to recombine with the HRP-ferryl to make O₂. The reasons for this are unknown; however, the pH dependence of the HRP reactions with chlorite may lend clues. Compound I formation from Fe(III) HRP and ClO^– or ClO ⁻₂ , but not ClO₂, was highly dependent on pH. In keeping with HRP’s known preference for neutral ligands, including peroxide [141], the neutral/protonated species (HClO, HClO₂) reacted preferentially with the Fe(III) iron. The distal histidine in HRP and all the well-studied plant peroxidases is critical for H₂O₂ deprotonation to form the Fe(III)-OOH (Compound 0) complex. It may similarly be involved in deprotonating chlorous acid (HClO₂, pK _a = 1.72) [20, 123]. By contrast, Clds lack a distal histidine and strongly prefer to bind cyanide and peracids in their anionic forms, with the distal arginine helping to secure the bound anion [78, 89]. The subsequent reactions of HRP-Compounds I and II with chlorite were also highly pH-dependent. The plotted dependencies of log k versus pH were once again linear with a slope of −1, indicating that Compounds I and II preferentially react with the HClO₂ acid rather than ClO ⁻₂ , in spite of the relative scarcity of the acid form (pK _a 1.72) in biochemically obtainable pH ranges. Again in contrast, it has been proposed that Compound I in Clds reacts with hypochlorite in its anionic form, in part because ClO⁻ is a better nucleophile than the HClO acid (pK _a 7.5), and in part because the steady state reaction is faster at low pH, where the distal arginine is expected to point toward the heme plane and to stabilize the ClO⁻ leaving group [78, 98]. The Clds’ ability to stabilize the hypochlorite leaving group in its anionic form may be critical for promoting O–O bond formation, and for distinguishing Clds from (O)Cl–O⁻ bond-cleaving, Compound I-forming peroxidases that nonetheless do not make O₂, such as HRP.

Alternatively or in addition, peroxidases are conventionally understood to react with their substrates on the periphery of the heme rather than at the apex of the distal pocket [121, 142–144]. Direct reaction of the porphyrin cation radical and substrate prevents cytochrome-P450-like oxygen-atom transfer from the Compound I intermediate to the substrate, which occurs when the substrate is able to approach the ferryl oxygen from an optimal angle. In Clds, reaction of hypochlorite with Compound I requires access to the same ferryl oxygen atom, while peroxidase-style one-electron reductions can just as well occur at the heme edge. It is possible that, in HRP, the distal pocket is not sterically configured to promote attack of the ClO⁻/HClO leaving group on the ferryl oxygen. Hence, O₂ production is not observed.

6 General Conclusions

As work with Clds illustrates, there is seemingly nothing that nature can’t do with a protein and a heme. The unusual transformation of ClO ⁻₂ to O₂ and Cl⁻ is greatly facilitated by enclosing heme in the relatively hydrophobic environment of chlorite dismutase, with a well-positioned arginine needed to recruit and steer the anionic substrate and intermediates. The arginine is a key innovation in an active site pocket that seems otherwise well-suited for little more than heme decomposition. The etiology of chlorite decomposition by Clds, the history of this fascinating protein family, and the biogeochemical consequences of natural perchlorate respiration all remain to be discovered by ongoing and future work.

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Acknowledgments

Support for this work from the National Institutes of Health, National Institute for General Medical Sciences is gratefully acknowledged (GM090260), as are the scientific and intellectual contributions of laboratory members past and present.

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Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717, USA
Jennifer L. DuBois & Sunil Ojha

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Fachbereich Biologie, Universita¨t Konstanz, Konstanz, Germany
Peter M. H Kroneck
Facultad de Química Departamento de Química Inorganica y Nuc, Universidad Nacional Autónoma de México, Ciudad de México, Distrito Federal, Mexico
Martha E. Sosa Torres

Abbreviations and Definitions

Cld:: chlorite dismutase
ClO^– :: hypochlorite
ClO₂ :: chlorine dioxide
ClO ⁻₂ :: chlorite
ClO ⁻₃ :: chlorate
ClO ⁻₄ :: perchlorate
Clr:: chlorate reductase
Compound 0:: Fe(III)-anion complex
Compound I:: Fe(IV)porphyrin cation radical
Compound II:: Fe(IV)=O or Fe(IV)–OH
DaCld:: Dechloromonas aromatica chlorite dismutase
DMSO:: dimethylsulfoxide
DyP:: dye-decoloring peroxidase
HClO:: hypochlorous acid
HRP:: horseradish peroxidase
MCD:: monochlorodimedone
MGD:: molybdopterin guanine dinucleotide
Nar:: nitrate reductase
NHE:: normal hydrogen electrode
NPRB:: non-perchlorate-respiring bacteria
NwCld:: Nitrospira winogradskyi chlorite dismutase
ONOO⁻ :: peroxynitrite
OTf:: triflate
Pcr:: perchlorate reductase
(per)chlorate:: chlorate and/or perchlorate
PRB:: perchlorate-respiring bacteria
PSII:: Photosystem II
Ser:: selenate reductase
TDMImP:: tetrakis-5,10,15,20-(N,N-dimethylimidazolium)porphyrinato
TF₄TMAP:: 5,10,15,20-tetrakis(tetrafluro-N,N,N-trimethylanilinium)porphyrinato
WCL:: wet chemistry laboratory

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DuBois, J.L., Ojha, S. (2015). Production of Dioxygen in the Dark: Dismutases of Oxyanions. In: Kroneck, P., Sosa Torres, M. (eds) Sustaining Life on Planet Earth: Metalloenzymes Mastering Dioxygen and Other Chewy Gases. Metal Ions in Life Sciences, vol 15. Springer, Cham. https://doi.org/10.1007/978-3-319-12415-5_3

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DOI: https://doi.org/10.1007/978-3-319-12415-5_3
Published: 01 December 2014
Publisher Name: Springer, Cham
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Online ISBN: 978-3-319-12415-5
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Production of Dioxygen in the Dark: Dismutases of Oxyanions

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Syntrophic anaerobic photosynthesis via direct interspecies electron transfer

The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis

A mechanism for water splitting and oxygen production in photosynthesis

Keywords

1 Introduction

2 Geochemistry of the Oxochlorates

2.1 Oxochlorates as Respiratory Anions

2.2 Natural Abundance of Perchlorate on Earth

2.3 Atmospheric and Extraterrestrial Origins of Perchlorate

2.4 Perchlorate on Mars

3 Perchlorate Respiration

3.1 Diversity of Perchlorate-Respiring Microbes

3.2 Genetics and Genomics of Perchlorate-Respiring Microbes

3.3 (Per)chlorate Reductases

3.4 Chlorite Dismutases and Perchlorate Respiration

4 Oxygen Generation by Chlorite Dismutases

4.1 Structures

4.1.1 Primary Structures: Diversity and Hallmarks of O2 Generation

4.1.2 Heme-Binding Domain

4.1.3 Active Site

4.1.4 Tertiary Structures and Oligomerization States

4.2 Reactivity and Mechanism

4.2.1 Diversity of Reactions Catalyzed by Chlorite Dismutases

4.2.2 Possible Pathways for O–O Formation

4.2.3 Catalytic Efficiency

4.2.4 Reaction Intermediates

4.2.5 Structure-Activity Relationships: Highlights

4.2.6 Heme and Protein Stability in Diverse Chlorite Dismutase Family Proteins

5 Synthetic and Biochemical Models

5.1 Chlorite as Reagent with Related Synthetic Metalloporphyrins

5.2 Reactions of Chlorite with Horseradish Peroxidase: Implications for Chlorite Dismutases

6 General Conclusions

References

Acknowledgments

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4.1.1 Primary Structures: Diversity and Hallmarks of O₂ Generation