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The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO(2)(−)) to yield Cl(−) and O(2). The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO(2)(−)) with the evolution of O(2) (k(cat) = (2.0±0.2)×10(2) s(−1); k(cat)/K(M) = (1.2±0.2)×10(5) M(−1) s(−1) at pH 5.2). Stopped-flow studies of this BrO(2)(−) 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 O(2) 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 ClO(2)(−). 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 ClO(2)(−) by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO(2), which forms in preference to the analogous to AA• in the BrO(2)(−) reaction. Addition of ClO(−) to Cpd II did not yield O(2). 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 O(2) production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.

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 Fe(III)–NO(●) character of the {FeNO}(6) ground state. Cld {FeNO}(6) complexes undergo slow reductive nitrosylation to yield {FeNO}(7) complexes. The affects 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 N(NO), 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.

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.

The H(2)O(2)-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 H(2)O(2). Time-resolved EPR spectroscopy revealed that Tyr-145 formed a radical species within 30 s of the reaction of the enzyme–coproheme complex with H(2)O(2). 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 H(2)O(2), 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 Cβ–H bond was partly rate-limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H(2)O(2) intermediate and then from the propionate Cβ–H to Tyr-145(•).

O(2)-evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO(2)(−)) to O(2) 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 ClO(2)(−), albeit with a 10-fold lower activity and a lower turnover number compared to 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 K(D) for KpCld–CN(−) is two 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 k(off). The kinetics of cyanide binding to DaCld, DaCld(R183Q) and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a pK(a) ~ 7 in stabilizing complexes of anionic ligands, including substrate. The Fe–C stretching and FeCN bending modes of DaCld–CN(−) (ν(Fe-C), 441 cm(−1); δ(FeCN), 396 cm(−1)) and KpCld–CN(−) (ν(Fe-C), 441 cm(−1); δ(FeCN), 356 cm(−1)) 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.

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 d...

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.

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 6.8 M GdnHCl at 37 °C, the half-life was 5 h. 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.

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.

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 w...

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 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 β-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., We describe a series of diketiminate-supported FeNNFe complexes in the formal Fe0 oxidation state, which have two alkali metal cations (alkali metal cation = Na+, K+, Rb+, Cs+) bound side-on to the N2 ligand. Larger cations twist the core of the molecule, but the back-bonding is similar through the series. Computations on the FeNNFe core show that the orbital energies move in compensating directions upon twisting, explaining the curious independence of the core metrics with twisting.

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 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 indicate 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 reduces readily in the presence of dithionite. Raman frequencies of the FeCO distortions in WT, H136A, and Y235A HmuT–CO complexes provide further evidence for the axial ligand assignments. Additionally, the se 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.

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.

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 Cl− binding 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 by 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.

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.

Heme-containing chlorite dismutases (Clds) catalyze a highly unusual O–O bond forming reaction. The O–O cleaving reactions of hydrogen peroxide and peracetic acid (PAA) with the Cld from Dechloromonas aromatica (DaCld) were studied to better understand the Cl–O cleavage of the natural substrate and subsequent O–O bond formation. While reactions with H2O2 resulted in slow destruction of the heme, at acidic pH, heterolytic cleavage of the O–O bond of PAA cleanly yielded the ferryl porphyrin cation radical (Compound I). At alkaline pH, the reaction proceeds more rapidly and the first observed intermediate is a ferryl heme. Freezequench EPR confirmed that the latter has an uncoupled protein-based radical, indicating that Compound I is the first intermediate formed at all pH values and that radical migration is faster at alkaline pH. These results suggest by analogy that two-electron Cl–O bond cleavage to yield a ferryl-porphyrin cation radical is the most likely initial step in O–O bond formation from chlorite.

The chlorite dismutases (Clds) degrade ClO(2)(-) to O(2) and Cl(-) in perchlorate respiring bacteria, and they serve still poorly defined cellular roles in other diverse microbes. These proteins share 3 highly conserved Trp residues, W155, W156, and W227, on the proximal side of the heme. The Cld from Dechloromonas aromatica (DaCld) has been shown to form protein-based radicals in its reactions with ClO(2)(-) and peracetic acid. The roles of the conserved Trp residues in radical generation and in enzymatic function were assessed via spectroscopic and kinetic analysis of their Phe mutants. The W155F mutant was the most dramatically affected, appearing to lose the characteristic pentameric oligomerization state, secondary structure, and heme binding properties of the WT protein. The W156F mutant initially retains many features of the WT protein but over time acquires many of the features of W155F. Conversion to an inactive, heme-free form is accelerated by dilution, suggesting loss of the protein's pentameric state. Hence, both W155 and W156 are important for heme binding and maintenance of the protein's reactive pentameric structure. W227F by contrast retains many properties of the WT protein. Important differences are noted in the transient kinetic reactions with peracetic acid (PAA), where W227F appears to form an [Fe(IV)=O]-containing intermediate, which subsequently converts to an uncoupled [Fe(IV)=O + AA(+)˙] system in a [PAA]-dependent manner. This is in contrast to the peroxidase-like formation of [Fe(IV)=O] coupled to a porphyrin π-cation radical in the WT protein, which decays in a [PAA]-independent manner. These observations and the lack of redox protection for the heme in any of the Trp mutants suggests a tendency for protein radical formation in DaCld that is independent of any of these conserved active site residues.

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 KIE = 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.

© 2016, SBIC. 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.

Erratum in : J Biol Chem. 2013 Jan 25;288(4):2190.; International audience; The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Heme is emerging as a key player in the synchrony of circadian-coupled transcriptional regulation. Current evidence suggests that levels of circadian-linked transcription are regulated in response to both the availability of intracellular heme and heme-based sensing of carbon monoxide (CO) and possibly nitric oxide (NO). The protein CLOCK is central to the regulation and maintenance of circadian rhythms in mammals. CLOCK comprises two PAS domains, each with a heme binding site. Our studies focus on the functionality of the murine CLOCK PAS-A domain (residues 103-265). We show that CLOCK PAS-A binds iron(III) protoporhyrin IX to form a complex with 1:1 stoichiometry. Optical absorbance and resonance Raman studies reveal that the heme of ferric CLOCK PAS-A is a six-coordinate, low-spin complex whose resonance Raman signature is insensitive to pH over the range of protein stability. Ferrous CLOCK PAS-A is a mixture of five-coordinate, high-spin and six-coordinate, low-spin complexes. Ferrous CLOCK PAS-A forms complexes with CO and NO. Ferric CLOCK PAS-A undergoes reductive nitrosylation in the presence of NO to generate a CLOCK PAS-A-NO, which is a five-coordinate {FeNO}(7) complex. Formation of the highly stable {FeNO}(7) heme complex from either ferrous or ferric heme makes possible the binding of NO at very low concentration, a characteristic of NO sensors. Comparison of the spectroscopic properties and CO-binding kinetics of CLOCK PAS-A with other CO sensor proteins reveals that CLOCK PAS-A exhibits chemical properties consistent with a heme-based gas sensor protein.

The uptake and utilization of heme as an iron source is a receptor-mediated process in bacterial pathogens and involves a number of proteins required for internalization and degradation of heme. In the following report we provide the first in-depth spectroscopic and functional characterization of a cytoplasmic heme-binding protein PhuS from the opportunistic pathogen Pseudomonas aeruginosa. Spectroscopic characterization of the heme-PhuS complex at neutral pH indicates that the heme is predominantly six-coordinate low spin. However, the resonance Raman spectra and global fit analysis of the UV-visible spectra show that at all pH values between 6 and 10 three distinct species are present to varying degrees. The distribution of the heme across multiple spin states and coordination number highlights the flexibility of the heme environment. We provide further evidence that the cytoplasmic heme-binding proteins, contrary to previous reports, are not heme oxygenases. The degradation of the heme-PhuS complex in the presence of a reducing agent is a result of H2O2 formed by direct reduction of molecular oxygen and does not yield biliverdin. In contrast, the heme-PhuS complex is an intracellular heme trafficking protein that specifically transfers heme to the previously characterized iron-regulated heme oxygenase pa-HO. Surface plasmon resonance experiments confirm that the transfer of heme is driven by a specific protein-protein interaction. This data taken together with the spectroscopic characterization is consistent with a protein that functions to shuttle heme within the cell.

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique in that heme is also required for maximal activity, although the function of heme in this enzyme is presently unclear. The study presented herein reveals that the heme of human CBS undergoes a coordination change upon reduction at elevated temperatures. We have termed this new species "CBS424" and demonstrate that its formation is likely irreversible when pH 9 Fe(III) CBS is reduced at moderately elevated temperatures (approximately 40 degrees C and higher) or when pH 9 Fe(II) CBS is heated to similar temperatures. Spectroscopic techniques, including resonance Raman, electronic absorption, and variable temperature/variable field magnetic circular dichroism spectroscopy, provide strong evidence that CBS424 is coordinated by two neutral donor ligands. It appears likely that the native cysteine(thiolate) heme ligand is displaced by an endogenous neutral donor upon conversion to CBS424. This behavior is consistent with other six-coordinate, cysteine(thiolate)-ligated heme centers, which seek to avoid this coordination structure in the Fe(II) state. Functional assays show that CBS424 is inactive and suggest that the ligand switch is responsible for eliminating enzyme activity. When this investigation is taken together with other functional studies of CBS, it provides strong evidence that coordination of Cys52 to the heme iron is crucial for full activity in this enzyme. We hypothesize that cysteine displacement may serve as a mechanism for CBS inactivation and that second-sphere interactions of the Cys52 thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site.

Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.

International audience; The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and chrome b5 reductase cytodomains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = approximately 0.05 and approximately 1 s(-1), respectively. The redox potential at the heme center of NCB5OR is -108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse.

This report presents a spectroscopic investigation of the nitrosyl adducts of FixL, the sensor in the signal transduction system responsible for regulating nitrogen fixation in Rhizobium meliloti. Variable-temperature resonance Raman (RR), electron spin resonance (ESR), and variable-temperature UV-visible absorption data are presented for the ferrous NO adducts of two FixL deletion derivatives, FixLN (the heme-containing domain) and FixL* (a functional heme-kinase). A temperature-dependent equilibrium is observed between the five-coordinate (5-c) and six-coordinate (6-c) ferrous nitrosyl adducts, with lower temperatures favoring formation of the 6-c nitrosyl adduct. This equilibrium is perturbed as the solution freezes, and the amount of 5-c FixL-NO increases sharply until a nearly constant ratio of 6-c to 5-c adducts is obtained. Complexation between the heme domain of FixL and its response regulator, FixJ, is revealed through specfic FixJ-induced increase in the energy separation between 5-c and 6-c FixL-NO. Ferric nitrosyl adducts of FixL* and FixLN autoreduce to their corresponding ferrous nitrosyl adducts. The kinetic behavior of this reduction is monophasic for FixL*-NO, while the reaction for ferric FixLN-NO is biphasic. These results suggest conformational inhomogeneity in the heme pocket of FixLN and conformational homogeneity in that of FixL*. Hence the kinase domain plays a role in distal pocket conformational stability. Implications for the signal transduction mechanism are discussed.

Vibrational assignments for the Fe-OH unit of ferric alkaline forms of two deletion derivatives of Rhizobium meliloti FixL, FixL*, a functional O2-sensing heme kinase, and FixLN, which contains only the heme domain, are made. Appearance of 2H- and 18O-sensitive Raman bands indicates that the heme group of FixL binds hydroxide as a distal ligand to form a six-coordinate complex. The alkaline FixLs are distributed between high- and low-spin states. The high- and low-spin bands corresponding to the ν (Fe-OH) modes occur at 479 and 539 cm–1, respectively. Low temperature favors formation of the low-spin complex, indicative of a thermal spin-state equilibrium. The ν (Fe-OH) frequencies of FixLN and FixL* are 11 to 18 cm–1 lower than those observed for the respective vibrations in alkaline myoglobin and hemoglobin. The weaker Fe-OH bond in the FixLs is attributed to a lack of hydrogen bonding on the distal side of the heme pocket.

For many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood.An alternative protocol for the production and purification of the outer membrane hemoglobin receptor (HmbR) from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques.HmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species. Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. Four HmbR variants with a modest decrease in binding efficiency for heme have been identified (H87C, H280A, Y282A, and Y456C). These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand.In summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidis, and suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter.A detailed understanding of the nutrient acquisition pathways in common pathogens such as N. meningitidis provides a foundation for new antimicrobial strategies.

FixL is a multiple-domain bacterial O2-sensing protein that modulates the activity of its kinase domain in response to O2 concentration. The kinase activity is coupled, via phosphoryl transfer, to transcriptional activation by a response-regulating protein, FixJ. Heme ligation resulting in a transition from high to low spin inhibits the kinase through an, as yet, ill-defined mechanism. This report presents spectroscopic, kinetic, and thermodynamic data on various complexes of two deletion derivatives of Rhizobium meliloti FixL, FixLN (the heme domain) and a functional heme kinase, FixL*. Resonance Raman characterization of metFixLN and metFixL* indicates that the heme core is smaller than that observed in metmyoglobin and is indicative of a five-coordinate high-spin heme in metFixLs. Resonance Raman spectra of FixL-CO adducts reveal that the Fe-C = O unit and/or its electrostatic environment in FixL*-CO is distorted relative to that in FixLN-CO. The 1H NMR spectra of the met forms further support the model of an asymmetric perturbation of the heme pocket structure associated with the presence of the kinase domain in FixL*. Observation of equivalent Fe-imidazole stretching vibrations for deoxyFixLN and deoxyFixL* (212 cm-1) indicates that the source of this perturbation in the heme pocket of FixL* does not lie on the proximal side of the heme. The equivalent Fe-imidazole stretching frequencies for deoxyFixLN and FixL* indicate that the presence of the kinase domain does not alter the relative strength of the proximal Fe-imidazole bond and that the proximal imidazole ligand is weakly H-bonded, probably to a backbone carbonyl group. Kinetic and thermodynamic data for the reactions of cyanide and fluoride ions with FixL are consistent with shape selectivity due to steric and/or an anisotropic electrostatic field in the distal heme pocket being responsible for the unique reactivities (or lack thereof) of FixL with ligands, i.e., O2, CO, CN-, F-, N3-, and SCN-. While the rate constants for binding of CN- to metFixLN and metFixL* are an order of magnitude slower than that for metMb, the stabilities of these complexes and metMb-CN are nearly the same. Neither N3- nor SCN- binds to the heme with measurable affinity. Since other ferric heme proteins form stable adducts with these ligands, the inability of FixL to form analogous complexes suggests that the ligand selectivity of this protein is rooted in insurmountable activation barriers to the binding of ligands containing more than two atoms and for ligands whose lowest-energy coordination geometries are linear. This allows the natural O2 ligand to compete kinetically with other naturally occurring ligands that form stable complexes with unencumbered hemes. Moreover, the rate constant for binding of CN- to the functional heme-kinase (metFixL*) is smaller than its metFixLN counterpart and the stability of metFixL*-CN is measurably lower than that of metFixLN-CN. This indicates that the contacts between the heme and kinase domains of FixL* impose more stringent geometric constraints on ligand binding than FixLN. The kinase is thus implicated in a possible mechanism for phosphate-dependent feedback control over ligand affinity of the heme.

The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O(2) and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O-O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV-visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pK(a) values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pK(a) of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid-base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b(5), localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b(5) reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.

Chlorite dismutase catalyzes O(2) release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady-state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3-11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid-base transition was observed by UV/visible and resonance Raman (rR) spectroscopy with a pK(a) of 8.7, 2-3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five-coordinate high-spin Fe(III) to a mixed high/low-spin ferric hydroxide, as confirmed by rR spectroscopy. The two Fe-OH stretching frequencies are quite low, consistent with a weak Fe-OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe-OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond-donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pK(a) approximately 6.5) form also appears to act as the active-site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady-state profile for the rate of chlorite decomposition is characterized by these same pK(a) values. The five-coordinate high-spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum k(cat) = 2.0(+/-0.6) x 10(5) s(-1), k(cat)/K(M) = 3.2(+/-0.4) x 10(7) M(-1) s(-1), pH 5.2, 4 degrees C) and to a somewhat lesser extent when it acts as a H-bond donor to the axial hydroxide ligand under alkaline conditions.

The oxygen-sensing FixL protein from Sinorhizobium meliloti is part of the heme-PAS family of gas sensors that regulate many important signal transduction pathways in a wide variety of organisms. We examined the role of the conserved F α -9 arginine 200 and several other conserved residues on the proximal F α -helix in the heme domain of Sm FixL* using site-directed mutagenesis in conjunction with UV–visible, EPR, and resonance Raman spectroscopy. The F α -helix variants R200A, E, Q, H, Y197A, and D195A were expressed at reasonable levels and purified to homogeneity. The R200I and Y201A variants did not express in observable quantities. Tyrosine 201 is crucial for forming the native protein fold of Sm FixL* while Y197 and R200 are important for stabilizing the kinase-inhibited oxy state. Our results show a clear correlation between H-bond donor ability of the F α -9 side chain and the rate of heme autoxidation. This trend in conjunction with crystal structures of liganded Bj FixL heme domains, show that H-bonding between the conserved F α -9 arginine and the heme-6-propionate group contributes to the kinetic stability of the kinase-inactivated, oxy state of Sm FixL*.

International audience; Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.

International audience; HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.

FixL proteins are bacterial heme-containing signal transduction proteins responsible for sensing the O2 concentration in the organism's environment. In Sinorhizobium meliloti, FixL is a protein histidine kinase that, together with its response regulator FixJ, constitute an oxygen-sensitive switch for regulation of the organism's nitrogen fixation and microaerobic respiration genes. The O2 sensitivity of the switch is such that it transitions during the process of symbiosis in alfalfa roots. Bradyrhizobium japonicum FixL similarly regulates microaerobic and anaerobic respiration genes during symbiosis in soybean roots. FixLs responds to low oxygen concentrations with increased autophosphorylation activity of their kinase domains. The phosphorylated FixL provides a phosphoryl group to FixJ within a FixLJ complex. The phosphorylated FixJs are transcriptionally active toward their target genes. The FixL kinase domain is inhibited when the heme in FixL is oxygenated. Kinetic and thermodynamic studies of ligand binding to both ferrous and ferric FixLs have shown a generally low affinity for ligands relative to myoglobins. These relatively low ligand affinities are attributable almost completely to diminished rates of ligand binding. The heme and its environment in liganded and unliganded FixLs have been characterized by UV-visible spectroscopy, resonance Raman spectroscopy, EXAFS, and X-ray crystallography. These studies have revealed that in the purified proteins, the heme is converted from a six-coordinate low spin state to a five-coordinate high spin state upon O2 release. Comparisons of spectroscopic and structural characteristics of deoxyFixL with oxy-FixL, met-FixL–CN, FixL–CO, and FixL–NO complexes indicate that distal affects in the heme pocket are, at least in part, responsible for communicating the ligation state of the heme to the kinase domain. The mechanisms by which ligand binding events are communicated from the heme to the kinase domain involve propagation and/or amplification of the ligation-coupled conformational transitions of the heme and its immediate protein environment. More recently, time-resolved experiments examining the non-equilibrium, ligand-coupled dynamics initiated by O2, CO, and NO photolysis from the corresponding FixL complexes have begun to shed light on the landscape of the switching coordinate. Site specific mutation of a number of amino-acid residues in the region of the heme environment have also provided valuable insight into the initial stages of the signal transduction event. Current thinking and understanding of the mechanism for signal transduction in the FixLJ systems are discussed in the context of these physical investigations.

PhuS is a cytoplasmic, 39 kDa heme-binding protein from Pseudomonas aeruginosa. It has previously been shown to transfer heme to its cognate heme oxygenase. It is expressed from the phu operon, which encodes a group of proteins known to actively internalize and transport heme from host organisms. This study combines the spectral resolution of resonance Raman spectroscopy with site-directed mutagenesis to identify and characterize the heme-bound states of holo-PhuS. This combined approach has identified a site in monomeric PhuS having alternate His ligands at positions 209 and 212. A second distinct binding site is present in dimeric PhuS. This site supports six-coordinate, low-spin heme, even when both His209 and His212 are mutated to Ala. The presence of conserved His and Tyr residues in all of the homologs characterized to date suggest that the dimer could be of the domain-swapped type in which two protein molecules are cross-linked by bound heme. The multiple heme-bound states and their sensitivity to pH suggest the possibility that these cytoplasmic heme-binding proteins have multiple functions that are toggled by variations in intracellular conditions.

We describe detailed studies of peptide-sandwiched mesohemes PSMA and PSMW, which comprise two histidine (His)-containing peptides covalently attached to the propionate groups of iron mesoporphyrin II. Some of the energy produced by ligation of the His side chains to Fe in the PSMs is invested in inducing helical conformations in the peptides. Replacing an alanine residue in each peptide of PSMA with tryptophan (Trp) to give PSMW generates additional energy via Trp side chain-porphyrin interactions, which enhances the peptide helicity and stability of the His-ligated state. The structural change strengthened His-FeIII ligation to a greater extent than His-FeII ligation, leading to a 56-mV negative shift in the midpoint reduction potential at pH 8 (Em,8 value). This is intriguing because converting PSMA to PSMW decreased heme solvent exposure, which would normally be expected to stabilize FeII relative to FeIII. This and other results presented herein suggest that differences in stability may be at least as important as differences in porphyrin solvent exposure in governing redox potentials of heme protein variants having identical heme ligation motifs. Support for this possibility is provided by the results of studies from our laboratories comparing the microsomal and mitochondrial isoforms of mammalian cytochrome b5. Our studies of the PSMs also revealed that reduction of FeIII to FeII reversed the relative affinities of the first and second His ligands for Fe (K2IIIK1III; K2IIK1II). We propose that this is a consequence of conformational mobility of the peptide components, coupled with the much greater ease with which FeII can be pulled from the mean plane of a porphyrin. An interesting consequence of this phenomenon, which we refer to as "dynamic strain", is that an exogenous ligand can compete with one of the His ligands in an FeII-PSM, a reaction accompanied by peptide helix unwinding. In this regard, the PSMs are better models of neuroglobin, CooA, and other six-coordinate ligand-sensing heme proteins than of stably bis(His)-ligated electron-transfer heme proteins such as cytochrome b5. Exclusive binding of exogenous ligands by the FeII form of PSMA led to positive shifts in its Em,8 value, which increases with increasing ligand strength. The possible relevance of this observation to the function of six-coordinate ligand-sensing heme proteins is discussed.

International audience; HasA is an extracellular heme binding protein, and HasR is an outer membrane receptor protein from Serratia marcescens. They are the initial partners of a heme internalization system allowing S. marcescens to scavenge heme at very low concentrations due to the very high affinity of HasA for heme (Ka = 5,3 x 10(10) m(-1)). Heme is then transferred to HasR, which has a lower affinity for heme. The mechanism of the heme transfer between HasA and HasR is largely unknown. HasR has been overexpressed and purified in holo and apo forms. It binds one heme molecule with a Ka of 5 x 10(6) m(-1) and shows the characteristic absorbance spectrum of a low spin heme iron. Both holoHasA and apoHasA bind tightly to apoHasR in a 1:1 stoichiometry. In this study we show that heme transfer occurs in vitro in the purified HasA.HasR complex, demonstrating that heme transfer is energy- and TonB complex-independent and driven by a protein-protein interaction. We also show that heme binding to HasR involves two conserved histidine residues.

FixL proteins are bacterial heme-containing signal transduction proteins responsible for sensing the O 2 concentration in the organism’s environment. In Sinorhizobium meliloti FixL is a protein histidine kinase that, together with its response regulator FixJ, constitute an oxygen-sensitive switch for regulation of the organism’s nitrogen fixation and microaerobic respiration genes. The O 2 sensitivity of the switch is such that it transitions during the process of symbiosis in alfalfa roots. Bradyrhizobium japonicum FixL similarly regulates microaerobic and anaerobic respiration genes during symbiosis in soybean roots. FixLs responds to low oxygen concentrations with increased autophosphorylation activity of their kinase domains. The phosphorylated FixL provides a phosphoryl group to FixJ within a FixLJ complex. The phosphorylated FixJs are transcriptionally active toward their target genes. The FixL kinase domain is inhibited when the heme in FixL is oxygenated. Kinetic and thermodynamic studies of ligand binding to both ferrous and ferric FixLs have shown a generally low affinity for ligands relative to myoglobins. These relatively low ligand affinities are attributable almost completely to diminished rates of ligand binding. The heme and its environment in liganded and unliganded FixLs have been characterized by UV–visible spectroscopy, resonance Raman spectroscopy, EXAFS, and X-ray crystallography. These studies have revealed that in the purified proteins, the heme is converted from a six-coordinate low spin state to a five-coordinate high spin state upon O 2 release. Comparisons of spectroscopic and structural characteristics of deoxyFixL with oxyFixL, met-FixL–CN, FixL–CO, and FixL–NO complexes indicate that distal affects in the heme pocket are, at least in part, responsible for communicating the ligation state of the heme to the kinase domain. The mechanisms by which ligand binding events are communicated from the heme to the kinase domain involves propagation and/or amplification of the ligation-coupled conformational transitions of the heme and its immediate protein environment. More recently, time-resolved experiments examining the nonequilibrium, ligand-coupled dynamics initiated by O 2 , CO, and NO photolysis from the corresponding FixL complexes have begun to shed light on the landscape of the switching coordinate. Current thinking and understanding of the mechanism for signal transduction in the FixLJ systems are discussed in the context of these physical investigations.

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli . In this work, native KatG and KatG(S315T) were purified from M. tuberculosis , and KatG(R463L) was purified from Mycobacterium bovis . The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K D for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.

Methionine-80 (Met-80) in mitochondrial cytochrome c (cyt c) can be oxidized to the corresponding sulfoxide by reactive oxygen species, a reaction of potential biological significance. As an approach to investigating how oxidation of Met-80 would influence its interactions with heme iron, we have examined binding of 2-(methylthio)ethanol (MTE) and dimethyl sulfoxide (DMSO), models for the side chains of Met and Met(SO), respectively, to ferrous and ferric N-acetylmicroperoxidase-8 (AcMP8). We find that DMSO coordinates 1.2 kcal/mol less strongly to Fe(III)-AcMP8 than does MTE, although both ligands form low-spin complexes. Comparison of spectroscopic data for the DMSO complex of Fe(III)-AcMP8 with published data for the Met(SO)-80 form of ferric cyt c allows us to conclude that Met(SO)-80 does not coordinate to iron in the latter. DMSO coordinates to Fe(II)-AcMP8 1.3 kcal/mol more strongly than does MTE, whereas Met-80 and Met(SO)-80 are reported to have approximately equal affinity for Fe(II) in cyt c. This result suggests that the steric environment near the heme iron in cyt c discriminates against coordination of Met(SO)-80. Vacuum quantum chemical density functional theory calculations confirm the greater affinity of the sulfoxide and show that coordination via oxygen is strongly favored. Resonance Raman spectroscopic data indicate that the preference for coordination via oxygen is maintained in solution. The computational data further indicate that the DMSO complex derives significant enthalpic stabilization from pi back-bonding but that iron to sulfur pi back-bonding does not make a significant contribution to bonding in the thioether complex.

This report presents evidence for interdomain steps of the ligand-coupled signal transduction mechanism of the oxygen receptor from Rhizobium meliloti, RmFixL. Photolysis of the CO adducts of heme domain (RmFixLN) and heme kinase (RmFixL*) proteins allowed tracking of second-order heme CO recombination reactions by transient absorbance. Whereas CO rebinding to RmFixLN is characterized by a single kinetic phase, rebinding to RmFixL* is characterized by two kinetic phases. Evidence indicates that CO rebinds to two interconvertible deoxyRmFixL* conformers that are produced sequentially after photolysis. Since the second conformer is only observed when the kinase domain is present, its production is concluded to be an interdomain signal transmission event that is coupled to heme ligand release. Because receptor clustering is a recurring theme in signal transduction mechanisms, the dependence of molecular weight upon heme ligation was investigated at equilibrium. Gel permeation chromatography and native gel electrophoresis showed that the molecular weight distribution for both RmFixLN and RmFixL* depends on heme ligation. At equilibrium, oxyRmFixLN and oxyRmFixL* exist as monomers and dimers, respectively. Their deoxy analogues, metRmFixLN and metRmFixL*, exist as dimers and as a mixture of tetramers and 9-mers, respectively. Assembly of these oligomers is reversible. The physiological relevance of these ligand-coupled assemblies and the kinetic factors controlling CO recombination are discussed.

KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid. About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T). The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes. Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts. Two conformers are observed for KatG-CO and KatG(S315T)-CO. Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue. The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit. Form II also has a neutral proximal histidine ligand but is not hydrogen bonded. This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band. The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum. Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO. Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO. Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers. Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.

The pH-dependent behavior of the ferric forms of two soluble truncations of Rhizobium meliloti FixL, FixL (heme and kinase domains, functional), and FixLN (heme domain) are examined by UV-visible, resonance Raman, and electron paramagnetic resonance spectroscopy. Global analysis of UV-visible data indicates that the pKa for hydroxide binding is slightly higher in FixL than in FixLN. Spectroscopic data show that high-spin and low-spin hydroxide adducts of FixLN and FixL exist in a thermal spin-state equilibrium with a significant fraction of the heme in the high spin form at room temperature. FixLN and FixL differ from myoglobin and hemoglobin in that their hemes are not fully ligated by hydroxide ion under strongly alkaline conditions. In addition to the binding of hydroxide ion, both FixLN and FixL undergo additional alkaline transitions that involve the deprotonation of tyrosine residues. FixLN contains four tyrosine residues. One has a pKa of 9.6, which is indistinguishable from that for hydroxide binding to the heme. The other three tyrosines have pKas greater than 11. At pH 11, the alkaline species react with cyanide to yield the familiar low-spin cyanide adduct. Upon reduction of the heme iron, the alkaline forms of the FixL deletion derivatives are converted to their deoxy forms. Resonance Raman spectra reveal that the Fe-His stretching vibrations of deoxyFixLN and deoxyFixL are not measurably shifted from those of their neutral counterparts. Treatment of the alkaline deoxyFixLs with O2 yields the respective oxy forms. Spectroscopic evidence indicates that the loss of activity at elevated pH cannot be attributed solely to generation of a low-spin heme hydroxide. Involvement of one or more tyrosines in signal transmission between the heme and kinase domains of FixL is proposed.

Resonance Raman spectra of the nitric oxide adducts of the ferrous forms of two soluble truncations of Rhizobium meliloti FixL, FixL* and FixLN, are reported. At room temperature, four isotope sensitive vibrations are observed for both ferrous FixL*-NO and ferrous FixLN-NO. For FixL*-NO, they are observed at 558, 525, 450, and 1675 cm(-1) and are assigned to v(Fe-NO) of a six-coordinate nitrosyl adduct, v(Fe-NO) of a five-coordinate nitrosyl adduct, delta(Fe-NO) of a six-coordinate nitrosyl adduct, and v(N-O) of a five-coordinate nitrosyl adduct, respectively. Similar frequencies are observed for the FixLN-NO isotope sensitive bands. On the basis of the frequencies and spectral separation of the v(Fe-NO) and delta(Fe-NO) modes, the Fe-N-O unit is concluded to have a bent geometry similar to those observed for the nitrosyl adducts of ferrous hemoglobin and myoglobin. Both proteins can be converted to predominantly five-coordinate nitrosyl adducts by lowering the temperature. In low-temperature resonance Raman spectra of FixL*-NO and FixLN-NO, the 558 cm(-1) bands are significantly decreased in intensity and v(Fe-NO)5-c (the Fe-NO stretching vibration for the five-coordinate nitrosyl adduct) is observed at 529 and 526 cm(-1), respectively. Analysis of the v3 and v8 vibrations for these nitrosyl adducts also supports the presence of both five- and six-coordinate nitrosyl adducts of FixL* and FixLN at room temperature and the conversion to predominantly five-coordinate nitrosyl adducts at low temperatures. This temperature-dependent interconversion is reversible. The possible physiological relevance of the thermally accessible five-coordinate state is discussed. The width of v(Fe-NO)6-c at half-height is 1.3 times broader in FixLN-NO than in FixL*-NO, suggesting that the Fe-N-O geometry is more homogeneous in FixL*-NO. In low-temperature spectra of FixLN-NO, a second v(N-O)5-c band is observed, indicating that more than one conformation is attainable in the five-coordinate FixLN-NO. This second v(N-O)5-c is not observed for five-coordinate FixL*-NO, further suggesting a more conformationally restricted nitrosyl heme in FixL*. These variations in the vibrations involving the Fe-NO unit indicate that the kinase domain influences the heme structure. The spectral differences are discussed in terms of the interdomain interactions that result in ligation-dependent mediation of the kinase activity.

This article discusses stepwise reduction of dinitrogen bond order by a low-coordinate iron complex.