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Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E degrees ') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the E degrees ' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature E degrees ' measurements. In this way, we have found that (i) the decrease in E degrees ' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetylmethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases E degrees ' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall E degrees ' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Although structure and steady-state kinetics of Clds have been elucidated, many questions remain (e.g., the mechanism of chlorite cleavage and the pH dependence of the reaction). Here, we present high-resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple, and the UV–vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite do not recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during turnover. Homolytic cleavage of chlorite is proposed.

Cytochromes c (cytc) are ubiquitous heme-containing metalloproteins that shuttle electrons in a variety of electron-transport chains, most often central to the production of the chemical energy necessary for cell life. The reduction potential (E°Ј) of the Fe 3+/2+ couple is central to the physiological role of these species in that it influences the thermodynamic and kinetic features of electron-exchange reactions with redox partners. In the last two decades, voltammetric techniques exploiting the heterogeneous electron exchange between cytc and solid electrodes have proved to be particularly valuable for the determination of E°Ј values for these species and for characterizing the mechanistic and kinetic aspects of the redox process for the various cytc conformers under a variety of solution conditions. The understanding of how, and to what extent, different molecular factors control the E°Ј value in these species has been the subject of much debate. First coordination sphere effects on the heme iron and the interactions of the heme group with the surrounding polypeptide [a] Gianantonio Battistuzzi obtained his Ph.D. in Chemistry from the University of Modena (Italy) in 1996, under the supervision of Prof. M. Sola, working on the purification and the characterization of the solution behavior of electron-transfer metalloproteins. After a period in the laboratories of Professors H. Witzel and B. Krebs at the University of Münster, he held a postdoctoral position at the University of Modena, where he was appointed Assistant Professor of Inorganic Chemistry in 1999. His main research interest lies in the understanding of the molecular and solution factors influencing the thermodynamics of the reduction of electron-transfer metalloproteins, in particular cytochromes c and blue copper proteins, through a combination of electrochemical and spectroscopic techniques. Marco Borsari obtained his Ph.D. in Chemistry from the University of Modena (Italy) in 1992, working with Prof. G. Gavioli on the characterization of the electrochemical behavior of sulfur-containing organic molecules and the determination of the nature and stability of metal complexes in solution through voltammetric and polarographic techniques. In 1992, he was appointed Assistant Professor of Physical Chemistry at the University of Modena and Reggio Emilia (Italy) and started working on the electrochemistry of metalloproteins with Marco Sola. He also established collaborations with the groups of I. Bertini and H. B. Gray. His main research activity has been directed towards the study of the redox properties of electron-transport proteins and redox enzymes, with a particular focus on the thermodynamics of the electron-transfer processes in cytochromes, blue copper, and ironϪsulfur proteins. Marco Sola obtained his Ph.D. in Chemistry from the University of Parma (Italy) in 1987. Between 1985 and 1991 he was a member of the group of I. Bertini and C. Luchinat in Florence working on NMR of paramagnetic metalloproteins. He spent 1988 as a research associate at ...

The thermodynamic parameters of protein reduction (∆H°′ rc and ∆S°′ rc ) were measured for a number of blue copper proteins including spinach plastocyanin, cucumber plastocyanin, Pseudomonas aeruginosa azurin, Rhus Vernicifera stellacyanin, cucumber stellacyanin, and horseradish umecyanin through voltammetric techniques in nonisothermal experiments at neutral pH. Including previous estimates for other members of the same protein family, we discuss here the thermodynamics of the electron-exchange reaction for twelve blue copper proteins from different sources. The enthalpic term (-∆H°′ rc /F) turns out to be the dominant contribution to the reduction potential in this protein class. However, the entropic term (T∆S°′ rc /F) heavily affects E°′, especially for the azurins. These data were analyzed in the light of the structural and dynamic information available on protein folding, geometric and electronic features of copper ligation, and solvation properties of the two redox states. It is clearly seen that the reduction enthalpy of the subfamily of the "phytocyanins" is less negative as compared to that of the other cupredoxins, most likely owing to a stronger axial ligation of the copper ion (which results in a nearly tetrahedral coordination geometry) and the greater exposition of the site to the solvent, which are both factors that stabilize the Cu(II) ion. The reduction entropy, which in most cases is negative, is instead apparently related to the solvation properties of the site. In addition, by analogy with class I cytochromes c, an increase in protein rigidity could also contribute to the entropy loss on reduction. Finally, it is apparent that the strategy of protein control of the reduction thermodynamics in high-potential electron-transfer metalloproteins (blue copper proteins, class I cytochromes c, HiPIPs) is the same: a dominant enthalpic term arising from ligand-binding interactions and electrostatic factors at the metal/protein interface, which strongly stabilizes the reduced state, is most often opposed by a weaker entropic term due to changes in protein dynamics and solvation properties, which disfavors protein reduction.

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

The M80A variant of yeast iso-1-cytochrome c (cytc), which features a noncoordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM) and investigated by cyclic voltammetry at varying conditions of temperature, pH, and O2 concentration. The E degrees ' value (standard reduction potential for the heme Fe(III)/Fe(II) couple) of M80A cytc on both SAMs is of approximately -200 mV (vs the standard hydrogen electrode, SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous electron transfer (ET) are dominated by the presence of a hydroxide ion as the sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is mainly responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range of 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.