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The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 ( C Cld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type C Cld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between C Cld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of C Cld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. This study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A, and D109A/E238A that could be recombinantly produced as apoproteins in Escherichia coli, fully reconstituted to the respective heme b proteins, and posttranslationally modified by hydrogen peroxide. This for the first time allows not only a direct comparison of the catalytic properties of the heme b and PTM forms but also a study of the impact of D109 and E238 on PTM and catalysis, including Compound I formation and the two-electron reduction of Compound I by bromide, iodide, and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238, in contrast to exchange of D109, does not cause significant structural rearrangements or changes in the catalytic properties neither in heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases.

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from "Candidatus Nitrospira defluvii" (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV-vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O═O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°' = -113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation.

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus "Nitrospira defluvii" (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The E°' values for free and cyanide-bound proteins were determined to be -119 and -397 mV for NwCld and -113 and -404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism.

The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.

Myeloperoxidase (MPO) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride to generate the potent bleaching hypochlorous acid, thus contributing to pathogen killing as well as inflammatory diseases. Its catalytic properties are closely related with unique posttranslational modifications of its prosthetic group. In MPO, modified heme b is covalently bound to the protein via two ester linkages and one sulfonium ion linkage with a strong impact on its (electronic) structure and biophysical and chemical properties. Here, the thermodynamics of the one-electron reduction of the ferric heme in wild-type recombinant MPO and variants with disrupted heme-protein bonds (M243V, E242Q, and D94V) have been investigated by thin-layer spectroelectrochemistry. It turns out that neither the oligomeric structure nor the N-terminal extension in recombinant MPO modifies the peculiar positive reduction potential (E°' = 0.001 V at 25 °C and pH 7.0) or the enthalpy or entropy of the Fe(III) to Fe(II) reduction. By contrast, upon disruption of the MPO-typical sulfonium ion linkage, the reduction potential is significantly lower (-0.182 V). The M243V mutant has an enthalpically stabilized ferric state, whereas its ferrous form is entropically favored because of the loss of rigidity of the distal H-bonding network. Exchange of an adjacent ester bond (E242Q) induced similar but less pronounced effects (E°' = -0.094 V), whereas in the D94V variant (E°' = -0.060 V), formation of the ferrous state is entropically disfavored. These findings are discussed with respect to the chlorination and bromination activity of the wild-type protein and the mutants., (© 2011 American Chemical Society)

Myeloperoxidase (MPO) (donor, hydrogen peroxide oxidoreductase, EC 1.11.1.7) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride (Cl-), to generate the potent bleaching oxidant hypochlorous acid (HOCl), thus contributing to bacterial killing and inflammatory reactions of neutrophils. Here, the thermodynamics of the one-electron reduction of the ferric heme in its ferric high-spin and cyanide-bound low-spin forms were determined through spectroelectrochemical experiments. The E(o)' values for free and cyanide-bound MPO (5 and -37 mV, respectively, at 25 degrees C and pH 7.0) are significantly higher than those of other heme peroxidases. Variable-temperature experiments revealed that the enthalpic stabilization of ferric high-spin MPO is much weaker than in other heme peroxidases and is exactly compensated by the entropic change upon reduction. In contrast to those of other heme peroxidases, the stabilization of the ferric cyanide-bound MPO is also very weak and fully entropic. This peculiar behavior is discussed with respect to the MPO-typical covalent heme to protein linkages as well as to the published structures of ferric MPO and its cyanide complex and the recently published structure of lactoperoxidase as well as the physiological role of MPO in bacterial killing.

Crystal structures and mass spectrometric analyses of catalase-peroxidases (KatGs) from different organisms revealed the existence of a peculiar distal Met-Tyr-Trp cross-link. The adduct appears to be important for the catalase but not the peroxidase activity of bifunctional KatG. To examine the effect of the adduct on enzyme redox properties and functions, we have determined the thermodynamics of ferric reduction for wild-type KatG and KatG(Y249F), whose tyrosine-to-phenylalanine mutation prevents cross-link formation. At 25 degrees C and pH 7.0, the reduction potential of wild-type KatG is found to be -226 +/- 10 mV, remarkably lower than the published literature values. The reduction potential of KatG(Y249F) is very similar (-222 +/- 10 mV), but variable temperature experiments revealed compensatory differences in reduction enthalpies and entropies. In both proteins, the oxidized state is enthalpically stabilized over the reduced state, but entropy is lost on reduction, which is in strong contrast to horseradish peroxidase, which also features a much more pronounced enthalpic stabilization of the ferriheme. With both proteins, the midpoint potential increased linearly with decreasing pH. We discuss whether the observed redox thermodynamics reflects the differences in structure and function between bifunctional KatG and monofunctional peroxidases.

The reduction thermodynamics (Delta H degrees '(rc) and Delta S degrees '(rc)) for native Paracoccus versutus amicyanin, for Alcaligenes faecalis S-6 pseudoazurin, and for the G45P, M64E, and K27C variants of Pseudomonas aeruginosa azurin were measured electrochemically. Comparison with the data available for other native and mutated blue copper proteins indicates that the features of metal coordination and the electrostatic potential due to the protein matrix and the solvent control the reduction enthalpy in a straightforward way. However, the effects on the reduction potential are rather unpredictable owing to the entropic contribution to E degrees ', which is mainly determined by solvent reorganization effects. Analysis of all the Delta H degrees '(rc) and Delta S degrees '(rc) values available for this protein class indicates that enthalpy-entropy compensation occurs in the reduction thermodynamics of wt cupredoxins from different sources, as well as for mutants of the same species. The findings indicate that the reduction enthalpies and entropies for these species are strongly affected by reduction-induced reorganization of solvent molecules within the solvation sphere of the protein. The absence of a perfect enthalpy-entropy compensation is due to the fact that while the differences between reduction entropies are dominated by solvent reorganization effects, those between reduction enthalpies are significantly controlled by intrinsic molecular factors related to the selective stabilization of the reduced form by coordination features of the copper site and electrostatic effects at the interface with the protein matrix.