Heme, the complex of iron and protoporphyrin IX, facilitates a litany of biochemical reactions, wherein its reactivity is directed by the protein or enzyme environment. Coproheme decarboxylase (ChdC, formerly known as HemQ) and chlorite dismutase (Cld) enzymes are closely related families within the CDE structural superfamily (pfam06778), [1] also called the peroxidase-chlorite dismutase superfamily. [2] While the folds and sequences of these enzymes are very similar, [3] differences in their active sites determine their unique functions based on cofactor specificity and reactivity.

ChdCs catalyze the hydrogen peroxide-dependent oxidative decarboxylation of coproheme III (hereinafter, coproheme) to yield heme b in the final step of the coproporphyrin-dependent heme b biosynthesis pathway in Gram-positive bacteria. [[4], [5], [6]] Each coproheme substrate undergoes two sequential catalytic transformations in which the iron porphyrinates serve as both catalytic cofactors and substrates. The propionate groups at β-pyrrole positions 2 and 4 of the coproheme macrocycle are thus converted to the vinyl groups of heme b. The first of these stepwise transformations occurs for propionate-2 yielding the half-product harderoheme III (hereinafter, harderoheme). This is followed by decarboxylation of propionate-4 to yield heme b. [4] The initial step in the catalytic model of the resting, ferric coproheme:ChdC reaction with H2O2, is formation of a high-valent, coproheme-centered intermediate, likely Compound I (FeIVO (por+•), Cpd I). That two-electron intermediate oxidizes the highly conserved Y145 to yield the corresponding tyrosyl radical (Y145•, numbering for ChdC from Staphylococcus aureus, SaChdC) and Compound II (FeIVO (por), Cpd II). The Y145• then abstracts a hydrogen atom from the μ-methylene carbon of the reactive propionate in a step that partially limits the overall reaction rate. [7] Electron transfer from the singly-oxidized propionate group to FeIVO then occurs with elimination of CO2 and formation of a new vinyl group. [7,8] Importantly, experimental evidence reveals that redox cycling of Y145 is required for both decarboxylations. Based on this evidence, it was proposed that harderoheme must reorient as a prelude to the second catalytic cycle to position propionate-4 close to Y145. [[7], [8], [9], [10]] Subsequently, this proposal was supported by computational and rR studies. [10,11]

The structural similarities between ChdCs and the Clds [12] warrants comparison of the structural bases of their functions and mechanisms. The Clds comprise two phylogenetically distinct clades that differ in their subunit size (∼32 kDa, clade 1; ∼21 kDa, clade 2). [1,13] The clade I Clds form pentamers analogous to those of ChdCs. Clds from both clades contain a catalytic heme b cofactor to decompose chlorite into O2 and Cl−. [[13], [14], [15], [16], [17], [18], [19], [20]] Several mechanisms for Cld-catalyzed degradation of chlorite have been proposed. First, the pentameric Cld from Dechloromonas aromatica RCB (DaCld) was proposed to form geminate Cpd I and OCl− followed by recombination to form an O−O bond in a FeIII peroxyhypochlorite complex as a prelude to product release. [16,[21], [22], [23], [24], [25]] An analogous mechanism was proposed for the dimeric clade 2 enzyme from Klebsiella pneumonia MGH 78578 Cld (KpCld) based on detection of HOCl upon its reaction with chlorite (ClO2−). [20] Subsequently, DFT calculations [26,27] and a kinetic study of the dimeric Cyanothece sp. PCC7425 Cld (CCld) ClO2− reaction [28] were interpreted as support for homolytic cleavage of the chlorite, generating ClO• and Cpd II as the catalytically relevant, high-valent intermediate. Rebinding of the ClO• would form a FeIII peroxyhypochlorite followed by released of Cl− and O2. Two additional mechanistic proposals suggested the involvement of protein-centered radicals in chlorite degradation. [29,30]

A recent mechanistic study of DaCld using bromite as an alternative O2-evolving substrate revealed that Cpd I is formed and only detectable during the production of O2. [31] It has since been reported that the CCld chlorite reaction also proceeds through Cpd I. [32] Hence, current experimental evidence supports a general Cld mechanism involving heterolytic O−Cl bond scission.

Comparison of ChdC with DaCld reveals some interesting similarities and differences. 1) Like ChdC, the DaCld reaction can produce a protein-based amino acid radical (AA•), hypothesized to be Y118•, which appears in tandem with Cpd II. Whereas the Cpd II:AA• state of DaCld is a catalytic dead-end, [31] the analogous Cpd II:Y145• state of SaChdC is required for catalysis. [25] 2) SaChdC binds its coproheme cofactor/substrate via a proximal histidine that has less imidazolate character than its counterpart in Clds. [16,[33], [34], [35]] 3) While the heme b and coproheme binding sites are in the same location within a tertiary subunit structure of DaCld and SaChdC, respectively, their porphyrin rings are rotated by 90° about the Fe-His bond. [12,36] 4) The conserved distal residue of both enzymes is observed in at least two H-bonding environments. A conserved distal Arg in Clds is catalytically important but not essential for chlorite decomposition. [24,37,38] In its open conformation, the Cld distal Arg is poised for chlorite recognition. [39] In its closed conformation, the distal Arg stabilizes complexes of anionic ligands like FeIII-ONO− and presumably FeIII-OClO− thereby inhibiting escape of chlorite degradation intermediates. [20,23] The conserved distal Gln in Mn-coproheme:ChdC from Geobacillus stearothermophilus (Gs) [36] is positioned above the coproheme with its side chain having two types of H-bonding environments (Fig. 1). In three subunits, Gln185 is H-bonded to the carboxylate of propionate-7 (P7). In the remaining subunits, Gln185 is part of a hydrogen bonding network involving a water molecule, Arg131 and the carboxylate of propionate-6 (P6). The difference in these two environments is the conformation of P6 and P7. The structure of coproheme:ChdC from Listeria monocytogenes (Lm) (PDB 6FXJ) [8] reveals variability in the distal Gln position within its five subunits. The distal Gln is within hydrogen bonding distance of P7 in two of the LmChdC subunits. This distribution of active site conformers is consistent with a dynamic catalytic site. 5) Comparison of the active sites of DaCld and GsChdC reveals that their respective distal Arg and Gln residues occupy similar positions above the metalloporphyrin plane. The nitrogen atoms of these two distal side chains that are closest to the iron are within 5.7 Å of that iron atom.

Here we examined the role played by the distal Gln in the decarboxylase and catalase activities of coproheme:SaChdC with its presumed physiological oxidant H2O2. The affinities for cyanide coproheme:SaChdC and distal variants Q185R and Q185A were determined. HCN is a frequently used probe for heme reactivity that is often compared with H2O2; both are neutral ligands whose binding to ferric heme occurs with loss of a proton. The stable ferric-cyano complex can be studied under equilibrium conditions in contrast to the ferric-hydroperoxo complex which typically undergoes further reaction. The distal steric and electrostatic landscapes of coproheme:SaChdCs were probed using cyanide, carbon monoxide, and hydroxide complexes. Vibrational characterization of the coproheme−CN− complexes allows for assessment of the steric environment of the bound cyanide, while the ferrous coproheme−CO and ferric coproheme−OH− reveal information about the ligand's H-bonding and electrostatic environment. These data revealed some similarities between coproheme:WT and SaChdC(Q185R) distal environments, and together, with chlorite dismutase activity assays, indicated that the distal environment of SaChdC does not support significant chlorite decomposition even with introduction of a distal Arg. Oxidative decarboxylation of coproheme with chlorite as the oxidant was readily observed and investigated with the three SaChdCs. Interestingly, characterization of the half product, harderoheme:SaChdC, was facilitated by its significant accumulation during the rapid coproheme:SaChdC reaction with ClO2−. Comparisons and contrasts in the roles of SaChdC's distal Gln with those of the conserved distal Arg in Clds are discussed.