Roles of High-valent Hemes and pH Dependence in Halite Decomposition Catalyzed by Chlorite Dismutase from Dechloromonas aromatica.

The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO2 -) to yield Cl- and O2. The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO2 -) with the evolution of O2 (k cat = (2.0±0.2)×102 s-1; k cat/K M = (1.2±0.2)×105 M-1 s-1 at pH 5.2). Stopped-flow studies of this BrO2 - decomposition as a function of pH show that 1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O2 evolution in acidic solution, 2) Cpd I and its one-electron reduction product, compound II (Cpd II) are present in varying ratios at intermediate pHs, and 3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a pK a of 6.7±0.1 in good agreement with the pK a of DaCld activity with ClO2 -. The observation of a protein-based amino acid radical (AA•) whose appearance coincides with that of Cpd II supports the hypothesis that conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having pK a 6.7. Examination of reaction mixtures comprising DaCld and ClO2 - by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO2, which forms in preference to the analogous to AA• in the BrO2 - reaction. Addition of ClO- to Cpd II did not yield O2. Together these results are consistent with heterolytic cleavage of the O-BrO- and O-ClO- bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O2 production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.

Structure and reactivity of chlorite dismutase nitrosyls.

Ferric nitrosyl ({FeNO}6) and ferrous nitrosyl ({FeNO}7) complexes of the chlorite dismutases (Cld) from Klebsiella pneumoniae and Dechloromonas aromatica have been characterized using UV-visible absorbance and Soret-excited resonance Raman spectroscopy. Both of these Clds form kinetically stable {FeNO}6 complexes and they occupy a unique region of ν(Fe-NO)/ν(N-O) correlation space for proximal histidine liganded heme proteins, characteristic of weak Fe-NO and N-O bonds. This location is attributed to admixed FeIII-NO character of the {FeNO}6 ground state. Cld {FeNO}6 complexes undergo slow reductive nitrosylation to yield {FeNO}7 complexes. The effects of proximal and distal environment on reductive nitroylsation rates for these dimeric and pentameric Clds are reported. The ν(Fe-NO) and ν(N-O) frequencies for Cld {FeNO}7 complexes reveal both six-coordinate (6c) and five-coordinate (5c) nitrosyl hemes. These 6c and 5c forms are in a pH dependent equilibrium. The 6c and 5c {FeNO}7 Cld frequencies provided positions of both Clds on their respective ν(Fe-NO) vs ν(N-O) correlation lines. The 6c {FeNO}7 complexes fall below (along the ν(Fe-NO) axis) the correlation line that reports hydrogen-bond donation to NNO, which is consistent with a relatively weak Fe-NO bond. Kinetic and spectroscopic evidence is consistent with the 5c {FeNO}7 Clds having NO coordinated on the proximal side of the heme, analogous to 5c {FeNO}7 hemes in proteins known to have NO sensing functions.

[(H2O)Zn(Imidazole)n]2+: the vital roles of coordination number and geometry in Zn-OH2 acidity and catalytic hydrolysis.

The Zn(ii)-(Imidazole(ate))n coordination motif occurs in numerous biochemical systems, including carbonic anhydrase and the matrix metalloproteinases (MMPs). Additionally, it has been used in synthetic materials, such as the zinc-based zeolitic imidazolate framework (ZIF) structures. Zinc centers in these systems typically act as Lewis acids that form complexes with small molecules, such as H2O, which is activated catalytically toward a number of important and useful hydrolysis reactions. The results reported herein from density functional theory (M05-2X) and ab initio (MP2 and CCSD(T)) calculations demonstrate that both the coordination number and the molecular geometry have a sizable impact on the binding strength, deprotonation energy, and acidity of the Zn(ii) coordinated water. Through a series of quantum mechanical calculations on [(ImH)nZn-OH2]2+ complexes (n = 1-5), both the solution-phase pKa and the gas-phase proton dissociation energy significantly increase as n increases. While this should not be too surprising, the Zn-OH2 bond dissociation energies and bond lengths don't necessarily undergo a concurrent decrease, and therefore would be of limited use as a prediction tool regarding Zn-OH2 acidity. In an effort to dissect the impacts of coordination number and molecular geometry on these thermodynamic parameters, we performed constrained geometry optimizations on the three- (n = 2) and four-coordinate (n = 3) complexes. These calculations surprisingly reveal a marked impact on the pKa and proton dissociation energy of the coordinated water, upon exclusive changes in the Zn(ii) coordination geometry, whether in the gas-phase or in aqueous solution. We discuss the relevance of these results to the catalytic peptide hydrolysis mechanism of the MMPs and possible implications for catalytic activity within or on the surfaces of ZIFs.

Effects of N2 Binding Mode on Iron-Based Functionalization of Dinitrogen to Form an Iron(III) Hydrazido Complex.

Distinguishing the reactivity differences between N2 complexes having different binding modes is crucial for the design of effective N2-functionalizing reactions. Here, we compare the reactions of a K-bridged, dinuclear FeNNFe complex with a monomeric Fe(N2) complex where the bimetallic core is broken up by the addition of chelating agents. The new anionic iron(0) dinitrogen complex has enhanced electron density at the distal N atoms of coordinated N2, and though the N2 is not as weakened in this monomeric compound, it is much more reactive toward silylation by (CH3)3SiI (TMSI). Double silylation of N2 gives a three-coordinate iron(III) hydrazido(2-) complex, which is finely balanced between coexisting S = 1/2 and S = 3/2 states that are characterized by crystallography, spectroscopy, and computations. These results give insight into the interdependence between binding modes, alkali dependence, reactivity, and magnetic properties within an iron system that functionalizes N2.

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b.

The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 formed a radical species within 30 s of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 s, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 s after initiating the reaction with H2O2, nor did it change the ∼270 s required for the radical signal to recede to ≤10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145• was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of ∼2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cß-H bond was partly rate-limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cß-H to Tyr-145•.

Distinguishing Active Site Characteristics of Chlorite Dismutases with Their Cyanide Complexes.

O2-evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO2-) to O2 and Cl-. Dechloromonas aromatica Cld ( DaCld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen Klebsiella pneumoniae contains a homodimeric Cld ( KpCld) that also decomposes ClO2-, albeit with an activity 10-fold lower and a turnover number lower than those of DaCld. The interactions between the distal pocket and heme ligand of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their cyanide complexes for insight into active site characteristics that are deterministic for chlorite decomposition. At 4.7 × 10-9 M, the KD for the KpCld-CN- complex is 2 orders of magnitude smaller than that of DaCld-CN- and indicates an affinity for CN- that is greater than that of most heme proteins. The difference in CN- affinity between Kp- and DaClds is predominantly due to differences in koff. The kinetics of binding of cyanide to DaCld, DaCld(R183Q), and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p Ka of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN- (νFe-C, 441 cm-1; δFeCN, 396 cm-1) and KpCld-CN- (νFe-C, 441 cm-1; δFeCN, 356 cm-1) complexes reveal differences in their FeCN angle, which suggest different distal pocket interactions with their bound cyanide. Conformational differences in their catalytic sites are also reported by the single ferrous KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.

Active Sites of O2-Evolving Chlorite Dismutases Probed by Halides and Hydroxides and New Iron-Ligand Vibrational Correlations.

O2-evolving chlorite dismutases (Clds) fall into two subfamilies, which efficiently convert ClO2- to O2 and Cl-. The Cld from Dechloromonas aromatica (DaCld) represents the chlorite-decomposing homopentameric enzymes found in perchlorate- and chlorate-respiring bacteria. The Cld from the Gram-negative human pathogen Klebsiella pneumoniae (KpCld) is representative of the second subfamily, comprising homodimeric enzymes having truncated N-termini. Here steric and nonbonding properties of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their fluorides, chlorides, and hydroxides. Cooperative binding of Cl- to KpCld drives formation of a hexacoordinate, high-spin aqua heme, whereas DaCld remains pentacoordinate and high-spin under analogous conditions. Fluoride coordinates to the heme iron in KpCld and DaCld, exhibiting ν(FeIII-F) bands at 385 and 390 cm-1, respectively. Correlation of these frequencies with their CT1 energies reveals strong H-bond donation to the F- ligand, indicating that atoms directly coordinated to heme iron are accessible to distal H-bond donation. New vibrational frequency correlations between either ν(FeIII-F) or ν(FeIII-OH) and ν(FeII-His) of Clds and other heme proteins are reported. These correlations orthogonalize proximal and distal effects on the bonding between iron and exogenous π-donor ligands. The axial Fe-X vibrations and the relationships between them illuminate both similarities and differences in the H-bonding and electrostatic properties of the distal and proximal heme environments in pentameric and dimeric Clds. Moreover, they provide general insight into the structural basis of reactivity toward substrates in heme-dependent enzymes and their mechanistic intermediates, especially those containing the ferryl moiety.

Enhancement of C-H Oxidizing Ability in Co-O2 †Complexes through an Isolated Heterobimetallic Oxo Intermediate.

The characterization of intermediates formed through the reaction of transition-metal complexes with dioxygen (O2 ) is important for understanding oxidation in biological and synthetic processes. Here, the reaction of the diketiminate-supported cobalt(I) complex LtBu Co with O2 gives a rare example of a side-on dioxygen complex of cobalt. Structural, spectroscopic, and computational data are most consistent with its assignment as a cobalt(III)-peroxo complex. Treatment of LtBu Co(O2 ) with low-valent Fe and Co diketiminate complexes affords isolable oxo species with M2 O2 "diamond" cores, including the first example of a crystallographically characterized heterobimetallic bis(µ-oxo) complex of two transition metals. The bimetallic species are capable of cleaving C-H bonds in the supporting ligands, and kinetic studies show that the Fe/Co heterobimetallic species activates C-H bonds much more rapidly than the Co/Co homobimetallic analogue. Thus heterobimetallic oxo intermediates provide a promising route for enhancing the rates of oxidation reactions.

Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae.

HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV-visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤5%) in comparison to the wild-type protein (~20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8M GdnHCl at 37°C, the half-life was 5h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.

Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ).

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H2O2 as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H2O2-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct C-H bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr•. This residue then abstracts a net hydrogen atom (H•) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO2 products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e-/H+ transfers and a novel route for the conversion of carboxylic acids to alkenes.

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b.

A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O2 and H2O2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O2 to the ferric state. The subsequent second-order reaction between the ferric complex and H2O2 is slow, pH-dependent, and further decelerated by D2O2 (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H2O2. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H2O2 cleavage is therefore unclear. From a cellular perspective, the use of H2O2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity.

The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein. Fe(II) DGCR8 RNA-binding heme domain (Rhed) undergoes a pH-dependent transition from 6-coordinate to 5-coordinate, due to protonation and loss of a lysine ligand; the ligand bound throughout the pH change is a histidine. Fe(II) Rhed binds CO and NO from 6- and 5-coordinate states, forming common CO and NO adducts at all pHs. Fe(II)-CO Rhed is 6-coordinate, low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO. Fe(II)-NO Rhed is 5-coordinate and pH insensitive. Fe(II)-NO also forms slowly upon reaction of Fe(III) Rhed with excess NO via a stepwise process. Heme reduction by NO is rate-limiting, and the rate would be negligible at physiological NO concentrations. Importantly, in vitro pri-miRNA processing assays show that both CO- and NO-bound DGCR8 species are inactive. Fe(II), Fe(II)-CO, and Fe(II)-NO Rhed do not bear either of the cysteine ligands found in the Fe(III) state. These data support a model in which the bis-cysteine thiolate ligand environment of Fe(III) DGCR8 is necessary for establishing proper pri-miRNA binding and enabling processing activity.

Corynebacterium diphtheriae HmuT: dissecting the roles of conserved residues in heme pocket stabilization.

The heme-binding protein HmuT is part of the Corynebacterium diphtheriae heme uptake pathway and is responsible for the delivery of heme to the HmuUV ABC transporter. HmuT binds heme with a conserved His/Tyr heme axial ligation motif. Sequence alignment revealed additional conserved residues of potential importance for heme binding: R237, Y272 and M292. In this study, site-directed mutations at these three positions provided insight into the nature of axial heme binding to the protein and its effect on the thermal stability of the heme-loaded protein fold. UV-visible absorbance, resonance Raman (rR) and thermal unfolding experiments, along with collision-induced dissociation electrospray ionization mass spectrometry, were used to probe the contributions of each mutated residue to the stability of ϖ HmuT. Thermal unfolding and rR experiments revealed that R237 and M292 are important residues for heme binding. Arginine 237 is a hydrogen-bond donor to the phenol side chain of Y235, which serves as an axial heme ligand. Methionine 292 serves a supporting structural role, favoring the R237 hydrogen-bond donation, which elicits a, heretofore, unobserved modulating influence on π donation by the axial tyrosine ligand in the heme carbonyl complex, HmuT-CO.

Alkali Metal Variation and Twisting of the FeNNFe Core in Bridging Diiron Dinitrogen Complexes.

Alkali metal cations can interact with Fe-N2 complexes, potentially enhancing back-bonding or influencing the geometry of the iron atom. These influences are relevant to large-scale N2 reduction by iron, such as in the FeMoco of nitrogenase and the alkali-promoted Haber-Bosch process. However, to our knowledge there have been no systematic studies of a large range of alkali metals regarding their influence on transition metal-dinitrogen complexes. In this work, we varied the alkali metal in [alkali cation]2[LFeNNFeL] complexes (L = bulky ß-diketiminate ligand) through the size range from Na(+) to K(+), Rb(+), and Cs(+). The FeNNFe cores have similar Fe-N and N-N distances and N-N stretching frequencies despite the drastic change in alkali metal cation size. The two diketiminates twist relative to one another, with larger dihedral angles accommodating the larger cations. In order to explain why the twisting has so little influence on the core, we performed density functional theory calculations on a simplified LFeNNFeL model, which show that the two metals surprisingly do not compete for back-bonding to the same π* orbital of N2, even when the ligand planes are parallel. This diiron system can tolerate distortion of the ligand planes through compensating orbital energy changes, and thus, a range of ligand orientations can give very similar energies.

Heme-bound SiaA from Streptococcus pyogenes: Effects of mutations and oxidation state on protein stability.

The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pKa values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wild-type; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61±3mV vs. SHE, similar to the wild-type protein (68±3mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process ranged from less than half a day to two days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release.

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.

The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

Methanethiol Binding Strengths and Deprotonation Energies in Zn(II)-Imidazole Complexes from M05-2X and MP2 Theories: Coordination Number and Geometry Influences Relevant to Zinc Enzymes.

Zn(II) is used in nature as a biocatalyst in hundreds of enzymes, and the structure and dynamics of its catalytic activity are subjects of considerable interest. Many of the Zn(II)-based enzymes are classified as hydrolytic enzymes, in which the Lewis acidic Zn(II) center facilitates proton transfer(s) to a Lewis base, from proton donors such as water or thiol. This report presents the results of a quantum computational study quantifying the dynamic relationship between the zinc coordination number (CN), its coordination geometry, and the thermodynamic driving force behind these proton transfers originating from a charge-neutral methylthiol ligand. Specifically, density functional theory (DFT) and second-order perturbation theory (MP2) calculations have been performed on a series of [(imidazole)nZn-S(H)CH3](2+) and [(imidazole)nZn-SCH3](+) complexes with the CN varied from 1 to 6, n = 0-5. As the number of imidazole ligands coordinated to zinc increases, the S-H proton dissociation energy also increases, (i.e., -S(H)CH3 becomes less acidic), and the Zn-S bond energy decreases. Furthermore, at a constant CN, the S-H proton dissociation energy decreases as the S-Zn-(ImH)n angles increase about their equilibrium position. The zinc-coordinated thiol can become more or less acidic depending upon the position of the coordinated imidazole ligands. The bonding and thermodynamic relationships discussed may apply to larger systems that utilize the [(His)3Zn(II)-L] complex as the catalytic site, including carbonic anhydrase, carboxypeptidase, ß-lactamase, the tumor necrosis factor-α-converting enzyme, and the matrix metalloproteinases.

Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ.

A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out.

A dimeric chlorite dismutase exhibits O2-generating activity and acts as a chlorite antioxidant in Klebsiella pneumoniae MGH 78578.

Chlorite dismutases (Clds) convert chlorite to O2 and Cl(-), stabilizing heme in the presence of strong oxidants and forming the O═O bond with high efficiency. The enzyme from the pathogen Klebsiella pneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite.

Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.