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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.

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.

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.

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.

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 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.

Distinguishing the reactivity differences between N(2) complexes having different binding modes is crucial for the design of effective N(2) functionalizing reactions. Here, we compare the reactions of a K-bridged, dinuclear FeNNFe complex with a monomeric Fe(N(2)) 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 N(2), and though the N(2) is not as weakened in this monomeric compound, it is much more reactive toward silylation by TMSI. Double silylation of N(2) 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 N(2).

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.

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, β-lactamase, the tumor necrosis factor-α-converting enzyme, and the matrix metalloproteinases.

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-carbonyl complexes are widely exploited for the insight they provide into the structural basis of function in heme-based proteins, by revealing the nature of their bonded and nonbonded interactions with the protein. This report presents two novel results which clearly establish a FeCO vibrational signature for crystallographically verified pentacoordination. First, anisotropy in the NRVS density of states for ν(Fe-C) and δ(FeCO) in oriented single crystals of [Fe(OEP)(CO)] clearly reveals that the Fe-C stretch occurs at higher frequency than the FeCO bend and considerably higher than any previously reported heme carbonyl. Second, DFT calculations on a series of heme carbonyls reveal that the frequency crossover occurs near the weak trans O atom donor, furan. As ν(Fe-C) occurs at lower frequencies than δ(FeCO) in all heme protein carbonyls reported to date, the results reported herein suggest that they are all hexacoordinate.

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.

Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a "cavity" is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low-spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (E(m,7) = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase.

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.

Understanding the interaction of N2 with iron is relevant to the iron catalyst used in the Haber process and to possible roles of the FeMoco active site of nitrogenase. The work reported here uses synthetic compounds to evaluate the extent of NN weakening in low-coordinate iron complexes with an FeNNFe core. The steric effects, oxidation level, presence of alkali metals, and coordination number of the iron atoms are varied, to gain insight into the factors that weaken the NN bond. Diiron complexes with a bridging N2 ligand, L(R)FeNNFeL(R) (L(R) = beta-diketiminate; R = Me, tBu), result from reduction of [L(R)FeCl]n under a dinitrogen atmosphere, and an iron(I) precursor of an N2 complex can be observed. X-ray crystallographic and resonance Raman data for L(R)FeNNFeL(R) show a reduction in the N-N bond order, and calculations (density functional and multireference) indicate that the bond weakening arises from cooperative back-bonding into the N2 pi orbitals. Increasing the coordination number of iron from three to four through binding of pyridines gives compounds with comparable N-N weakening, and both are substantially weakened relative to five-coordinate iron-N2 complexes, even those with a lower oxidation state. Treatment of L(R)FeNNFeL(R) with KC8 gives K2L(R)FeNNFeL(R), and calculations indicate that reduction of the iron and alkali metal coordination cooperatively weaken the N-N bond. The complexes L(R)FeNNFeL(R) react as iron(I) fragments, losing N2 to yield iron(I) phosphine, CO, and benzene complexes. They also reduce ketones and aldehydes to give the products of pinacol coupling. The K2L(R)FeNNFeL(R) compounds can be alkylated at iron, with loss of N2.

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.

A recent report of the structural and vibrational properties of heme-bound HNO in myoglobin, MbHNO, revealed a long Fe-N H N O bond with the hydrogen atom bonded to the coordinated N atom. The Fe-N(H)-O moiety was reported to exhibit an unusually high Fe-N H N O stretching frequency relative to those of the corresponding {FeNO} 6 and {FeNO} 7 porphyrinates, despite the Fe-N H N O bond being longer than either of its Fe-N N O counterparts. Herein, we present results from density functional theory calculations of an active site model of MbHNO that support the previous assignment and clarify this seemingly contradictory result. The results are consistent with the experimental evidence for a ground-state Fe-N(H)-O structure having a long Fe-N H N O bond and a uniquely high ν F e - N ( H N O ) frequency. This high frequency is the result of the correspondingly low reduced mass of the normal mode, which is largely attributable to significant motion of the N-bound hydrogen atom. Additionally, the calculations show the Fe-N(H)O bonding in this complex to be remarkably insensitive to whether the HNO and ImH ligand planes are parallel or perpendicular. This is attributed to insensitivities of the Fe-L a x i a l characters of molecular orbitals to the relative HNO and ImH orientation in both the parallel and perpendicular conformers.

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.

In this report, we present a quantum chemical/density functional theory (DFT) study of possible zinc binding modes for five imidazole- and thiol-based ligands relevant to metzincin (MMP and ADAM) inhibitor design. The gas-phase DFT calculations show that, while the imidazole-based ligands may bind zinc in either a five-coordinate bidentate or a four-coordinate monodentate manner, the deprotonated thiolate-containing mercaptoketone and mercapto alcohol ligands are not strongly bidentate, in agreement with recently reported model complex structures. On the basis of modeling of ligand−water exchange reactions, we estimate that the free energy released upon coordination of these zinc binding groups to metzincins decreases in the order mercaptoketone > mercapto alcohol > imidazole-based. The range of binding free energies, however, is only about a few kcal/mol, in the gas phase. In addition, calculated proton dissociation energies show that the driving force for deprotonation of coordinated thiol is greater th...

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.

Several tripodal diimine ligands, tris(2-(2-thiazolyl)methyliminoethyl)amine, 2-Tatren, tris(2-(4-(5-methyl)imidazolyl)methyliminoethyl)amine, 5-Me-4-Imtren, tris(2-(4-imidazolyl)methyliminoethyl)amine, 4-Imtren, tris(2-(2-imidazolyl)methyliminoethyl)amine, 2-Imtren, and their Mg(2+) complexes were prepared and characterized. X-ray diffraction studies show that the Mg(2+) ions are six-coordinate, with three acyclic imine N atoms and three imidazolyl or thiazolyl N atoms coordinated with the general formula [Mg(L)](ClO(4))(2) (L=4-Imtren (1), 2-Imtren (2), 2-Tatren (3), and 5-Me-4-Imtren (4)). These complexes are chiral with both Delta and Lambda isomers present in the unit cell. (1)H NMR titrations reveal that complexes also form in solution and that the chirality is maintained. Variable temperature (1)H NMR reveals that the Delta and Lambda isomers interconvert in the intermediate to slow time scale. The interconversion rate slows with increasing pK(a) of the ligand heterocycle, suggesting that interconversion proceeds through a partially dissociated state. These complexes undergo trans-metallation by Zn(2+), indicating that their ligands can be released in a kinetically facile manner to form more stable metal ion complexes.

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.

Resonance Raman spectra of dicopper complexes [L2Cu2(μ-O)2]2+ contain a number of resonance-enhanced features between 500 and 900 cm-1, with L = R3TACN and various bidentate ligands (TACN = 1,4,7-triazacyclononane). Most importantly, there is a vibration near 600 cm-1 in all [L2Cu2(μ-O)2]2+ compounds that is polarized, shifts by 19−27 cm-1 upon 18O2 substitution, and gives a new peak with 16O18O substitution, identifying it as a totally symmetric Ag vibration of the bis(μ-oxo)dicopper(III) core. Changing the pendant groups on R3TACN causes shifts in the frequency of the Cu2(μ-O)2 vibration, but the direction of these shifts depends on the details of the organic fragment. A substantial shift to higher frequency is evident when bidentate ligands are used in place of TACN. Importantly, bidentate ligands with two different types of nitrogen donors show two independent core vibrations; these are assigned as “breathing” and “pairwise” modes through simple group theory considerations as well as by calculations u...

Der direkte Angriff des Bis(μ-oxo)dikupfer-Kerns auf ein Aren scheint auf der Grundlage von Studien an neuen [CuIII2(μ-O)2]2 +-Verbindungen, die durch zweizahnige Imin/Amin-Liganden stabilisiert werden, in Tyrosinase und Modellkomplexen moglich zu sein. Anhand der Reaktivitat eines Bis(μ-oxo)dikupfer-Kerns wird gezeigt, das der Zerfall dieses Intermediates einen gebundenen Phenylring in einer Reaktion, die der durch Tyrosinase katalysierten analog ist, hydroxyliert [Gl. (a)].

Direct attack of the bis(μ-oxo)dicopper core on an arene appears feasible in tyrosinase and model complexes on the basis of studies of new [Cu(III) 2 (μ-O)2 ](2+) compounds supported by bidentate imine/amine ligands. In the first demonstration of such reactivity for a bis(μ-oxo)dicopper core, decomposition of these intermediates caused hydroxylation of a pendant phenyl ring [Eq. (a)] in a reaction analogous to that catalyzed by tyrosinase.

On the basis of static and time-resolved resonance Raman spectroscopy of HbA and of a mutant, HbK (Dalpha99N), a specific reaction coordinate is proposed for the allosteric transition in human hemoglobin. The heme is held between proximal (F) and distal (E) helices, whose orientation is responsive to forces generated by ligation and deligation. The E and F helices are in turn tethered via H-bonds to the A and H helices. These outer helices follow the E-F motion, thereby repositioning the N- and C-termini, which form the intersubunit salt bridges in the T quaternary structure. When the T state interface is weakened by Asp --Asn substitution at a quaternary H-bond (HbK), the Fe-His bond is relaxed and becomes responsive to allosteric effectors. The same E-F motion is observed in HbK, but the A-H following motion is delayed, relative to HbA, as is the Asn H-bond formation.

Isotope labeling has been used to assign the resonance Raman spectra of cytochrome c peroxidase, expressed in Escherichia coli [CCP (MKT)], and of the D235N site mutant. 54Fe labeling establishes the coexistence of two separate bands (233 and 246 cm-1), arising from the stretching of the bond between the Fe atom and the proximal histidine ligand, His175. These are assigned to tautomers of the H-bond between the His175 imidazole NΓH proton and the Asp235 carboxylate side chain: In one tautomer the proton resides on the imidazole while in the other the proton is transferred to the carboxylate. When Asp235 is replaced by Asn, the H-bond is lost, and the Fe-His stretching frequency is markedly lowered. Two new RR bands are produced, at 205 and 185 cm-1, as a result of coupling between the shifted Fe-His vibration and a nearby porphyrin mode; the two bands share the 54Fe sensitivity expected for Fe-His stretching. C=C stretching and CβC=C bending vibrations have been separately assigned to the 2- and 4-vinyl groups of the protoheme prosthetic group via selective vinyl deuteration. In the acid form of the enzyme, the frequencies coincide for the two vinyl groups, at 1618 cm-1 for the C=C stretch, and at 406 cm-1 for the CβC=C bend. However, the 2-vinyl frequencies are elevated in the alkaline form of the enzyme, to 1628 cm-1 for C=C stretching, and to 418 cm-1 for CβC=C bending, while the 4-vinyl frequencies remain unshifted. Thus, the acid-alkaline transition involves a protein conformation change that specifically perturbs the 2-vinyl substituent. This perturbation might be a reorientation of the vinyl group, or an alteration of the porphyrin geometry that affects the porphyrin-vinyl coupling. The perturbation is attenuated when CO is bound to the enzyme; the C=C frequency is then unaffected in the alkaline form, while the CβC=C bending frequency is shifted to a smaller extent (412 cm-1). This attenuation is probably linked to inhibition of distal histidine binding to the heme Fe in the alkaline form when the CO is bound. © 1996 John Wiley & Sons, Inc.

The synthesis and characterization of a series of meso-2-pyridylporphyrins and their Mg(2+) complexes are reported. Condensation of 4-alkylbenzyl-2,2'-dipyrromethanes (alkyl = Me, n-Pr, or n-Bu) with 2-pyridinecarboxaldehyde yielded a series of free-base meso-2-pyridylporphyrins. Insertion of Mg(2+) into the free-base porphyrins yielded the respective magnesium complexes. These compounds were characterized using 1D ((1)H and (13)C) and 2D ((1)H-(1)H COSY) NMR methods, UV-visible absorption spectroscopy, fluorescence spectroscopy, and mass spectrometry. The interplanar spacing of the dimers is sufficiently small that there is excitonic coupling of the constituent chromophores. The overall dissociation constant of these dimers is estimated at 2 x 10(-)(6) M. Addition of donor ligands such as acetone, DMF, DMSO, or pyridine converts the dimeric species to their respective constituent monomers. Titration of the dimeric complex with pyridine-d(5)() shows that disaggregation requires coordination of two pyridine molecules at independent binding sites. Tracking of the pyridine coordination by (1)H NMR spectroscopy allowed for determination of the equilibrium constant for the pyridine-induced disaggregation reaction (2.1 x 10(-)(3) M(2)). Both the spontaneous dissociation and the pyridine-induced disaggregation reactions occur by two steps.

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 end states, R and T, of the allosteric transition in hemoglobin (Hb) are structurally well characterized, but there is little information on intermediate structures along the allosteric pathway. These intermediates were examined by means of time-resolved resonance Raman spectroscopy in the nanosecond-to-microsecond interval after HbCO photolysis. Complementary spectra of the heme group and of the tyrosine and tryptophan residues were recorded during laser excitation at 436 and 230 nanometers. These spectra reveal a sequence of interleaved tertiary and quaternary motions during the photocycle, motions involving the proximal and distal helices, and the alpha 1 beta 2 subunit interface. This sequence leads to a modified form of the T state, in which the alpha 1 beta 2 interface is deformed as a result of two carbon monoxide molecules binding to the same dimer within the tetramer.

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.