Redox Potential Of Cytochrome Research Articles

Ab Initio Evaluation of the Redox Potential of Cytochrome c.

Various biochemical activities of metabolism and biosynthesis are fulfilled by redox processes with explicit electron exchange, which furnish redox enzymes with high chemical reactivity. However, theoretical investigation of a redox process, which simultaneously involves a complex electronic change at a redox metal center and conformational reorganization of the surrounding protein environment coupled to the electronic change, requires computationally conflicting approaches, highly accurate quantum chemical calculations, and long-time molecular dynamics (MD) simulations, limiting the physicochemical understanding of biological redox processes. Here, we theoretically examined a redox process of cytochrome c by means of a hybrid molecular simulation technique, which enables one to consistently treat the redox center at the ab initio quantum chemistry level of theory and the protein reorganization with long-time MD simulations on the microsecond timescale. The calculations successfully evaluated a large absolute redox potential, 4.34 eV, with errors of only 0.03 to 0.34 eV to the experimental ones without any problem-specific empirical parameters. Through the long-time MD sampling, large and nonlinear reorganization of the protein environment was unveiled and the molecular determinants for the redox potential were identified. The present ab initio approach significantly expands the applicability of theoretical investigation to biological redox systems with more electronically complicated redox centers such as polynuclear transition metal complexes.

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.

Microbial electrosynthesis of carboxylic acids through CO2 reduction with selectively enriched biocatalyst: Microbial dynamics

Microbial electrosynthesis is an emerging strategy of transforming CO2 into valuable chemicals by exploiting the capabilities of bacteria. As biocatalyst is considered as prime factor in bioelectrochemical CO2 reduction, we used an enrichment technique comprising of heat pretreatment of parent inoculum followed by enrichment under gas mixture of H2 and CO2. Here, we use a double chambered bioelectrochemical system (BESH) wherein cathode chamber was inoculated with enriched acetogenic (homoacetogenic) bacteria, subsequently polarized with voltage of −0.8V vs Ag/AgCl (s). BESH operation resulted in synthesis of carboxylic acids/volatile fatty acids (VFA), with major proportion confined to production of acetic acid (12.57mM) with a maximum current density of 650±50mA/m2 generated in response to applied voltage. While in the previous study, BESC (untreated) depicted comparatively low yield of acetic acid (3.19mM). A maximum of 67% reduction in bicarbonate concentration and columbic efficiency of 38% was noticed signifying the utilization of inorganic carbon for bio-electrochemical synthesis of acetic acid. Efficiency of BES was also discussed in terms of energy discharge. Few signals were obtained on voltammetric signature at various potentials which might correspond to the redox potential of cytochromes, flavo proteins and Fe-S proteins suggesting their involvement as electron carriers during microbial electrosynthesis. Phylogenetic analysis was comparatively made between untreated and heat treated enriched bacteria which revealed members belonging to Clostridiaceae dominant in heat treated inoculum, while methanogens appeared in untreated inoculum. Selective enrichment of biocatalyst proves to be advantageous for CO2 reduction in BES which yielded high productivity of carboxylic acids in comparison to untreated biocatalyst.

Measurement of the Mitochondrial Membrane Potential and pH Gradient from the Redox Poise of the Hemes of the bc1 Complex

The redox potentials of the hemes of the mitochondrial bc1 complex are dependent on the proton-motive force due to the energy transduction. This allows the membrane potential and pH gradient components to be calculated from the oxidation state of the hemes measured with multi-wavelength cell spectroscopy. Oxidation states were measured in living RAW 264.7 cells under varying electron flux and membrane potential obtained by a combination of oligomycin and titration with a proton ionophore. A stochastic model of bc1 turnover was used to confirm that the membrane potential and redox potential of the ubiquinone pool could be measured from the redox poise of the b-hemes under physiological conditions assuming the redox couples are in equilibrium. The pH gradient was then calculated from the difference in redox potentials of cytochrome c and ubiquinone pool using the stochastic model to evaluate the ΔG of the bc1 complex. The technique allows absolute quantification of the membrane potential, pH gradient, and proton-motive force without the need for genetic manipulation or exogenous compounds.

Do Tyrosine Phenolic Groups Contribute to the Alkaline Transition in the Redox Potential of Cytochrome F?

Cytochrome f, a c-type cytochrome involved in the photosynthetic electron transport chain, has a significantly higher redox potential than most other c-type cytochromes, ranging +370 to +380 mV. Like cytochrome c, cytochrome f also exhibits an alkaline transition in which the redox potential becomes pH dependent at high pH. In the case of cytochrome c, this has been attributed to replacement of the methionine sulfur serving as the sixth iron ligand by a deprotonated amino group. This cannot be the cause for the alkaline transition in cytochrome f as there is no methionine ligand to the iron, the sixth position being occupied by the N-terminal amino group.

Reversible increase in the redox potential of cytochrome c in methanol

The E degrees ' of cytochrome c on a self-assembled monolayer (SAM) modified gold electrode increased by 300 mV immediately on immersion in methanol; on re-immersion of the electrode in aqueous buffer, the original faradaic response was restored, but over a period of 120 min, indicating that methanol causes a significant change in conformation/orientation of the protein.

S13.8 Tuning the electron transfer rate by the redox potential of cytochrome c in complex with cytochrome c oxidase

S13.8 Tuning the electron transfer rate by the redox potential of cytochrome c in complex with cytochrome c oxidase

Spectroscopic Measurement of the Redox Potential of Cytochrome c for the Undergraduate Biochemistry Laboratory

Teaching redox chemistry is an important facet of undergraduate biochemistry courses, particularly with respect to developing the students' understanding of the central metabolic processes such as the mitochondrial respiratory chain and photosynthesis. We have introduced an experiment for the undergraduate metabolism course involving the spectroscopic measurement of the redox potential of cytochrome c. The experiment permits a quantitative result within a single three-hour laboratory period and complements the basic redox concepts and calculations shown in class.

Crystal structure of oxidized cytochrome c6A from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c 6, cytochrome c 6A from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c 6A from Arabidopsis thaliana at 1.5 Å resolution in order to help elucidate its function. The overall structure of cytochrome c 6A follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c 6A. Our model provides a chemical basis for the known low redox potential of cytochrome c 6A which makes it an unsuitable electron carrier between cytochrome b 6 f and PSI.

The Redox Chemistry of the Covalently Immobilized Native and Low-pH Forms of Yeast Iso-1-cytochrome c

Cyclic voltammetry experiments were carried out on native Saccharomyces cerevisiae iso-1-cytochrome c and its C102T/N62C variant immobilized on bare polycrystalline gold electrode through the S-Au bond formed by a surface cysteine. Experiments were carried out at different temperatures (5-65 degrees C) and pH values (1.5-7). The E degrees ' value at pH 7 (+370 mV vs SHE) is approximately 100 mV higher than that for the protein in solution. This difference is enthalpic in origin and is proposed to be the result of the electrostatic repulsion among the densely packed molecules onto the electrode surface. Two additional electrochemical waves are observed upon lowering the pH below 5 (E degrees ' = +182 mV) and 3 (E degrees ' = +71 mV), which are attributed to two conformers (referred to as "intermediate" and "acidic", respectively) featuring an altered heme axial ligation. This is the first determination of the reduction potential for low-pH conformers of cytochrome c in the absence of denaturants. Since the native form of cytochrome c can be restored, bringing back the pH to neutrality, the possibility offered by this transition to reversibly modulate the redox potential of cytochrome c is appealing for bioelectronic applications. The immobilized C102T/N62C variant, which differs from the native protein in the orientation of the heme group with respect to the electrode, shows very similar reduction thermodynamics. For both species, the rate constant for electron transfer between the heme and the electrode increases for the acidic conformer, which is also found to act as a biocatalytic interface for dioxygen reduction.

Cytochrome c and bioenergetic hypothetical model for alkaliphilic Bacillus spp.

Although a bioenergetic parameter is unfavorable for production of ATP (DeltapH<0), the growth rate and yield of alkaliphilic Bacillus strains are higher than those of neutralophilic Bacillus subtilis. This finding suggests that alkaliphiles possess a unique energy-producing machinery taking advantage of the alkaline environment. Expected bioenergetic parameters for the production of ATP (DeltapH and DeltaPsi) do not reflect the actual parameters for energy production. Certain strains of alkaliphilic Bacillus spp. possess large amounts of cytochrome c when grown at a high pH. The growth rate and yield are higher at pH 10 than at pH 7 in facultative alkaliphiles. These findings suggest that a large amount of cytochrome c at high pHs (e.g., pH 10) may be advantageous for sustaining growth. To date, isolated cytochromes c of alkaliphiles have a very low midpoint redox potential (less than +100 mV) compared with those of neutralophiles (approximately +220 mV). On the other hand, the redox potential of the electron acceptor from cytochrome c, that is, cytochrome c oxidase, seems to be normal (redox potential of cytochrome a=+250 mV). This large difference in midpoint redox potential between cytochrome c and cytochrome a concomitant with the configuration (e.g., a larger negative ion capacity at the inner surface membrane than at the outer surface for the attraction of H+ to the intracellular membrane and a large amount of cyrochrome c) supporting H+-coupled electron transfer of cytochrome c may have an important meaning in the adaptation of alkaliphiles at high pHs. This respiratory system includes a more rapid and efficient H+ and e- flow across the membrane in alkaliphiles than in neutralophiles.

Structure and Redox Properties of the Haem Centre in the C357M Mutant of Cytochrome P450cam

The effects of site-specific mutation of the axial cysteine (C357M) to a methionine residue in cytochrome P450cam on the enzyme's coordination geometry and redox potential have been investigated. The absorption spectra of the haem centre in the C357M mutant of the enzyme showed close similarity to those of cytochrome c both in the oxidised and reduced forms. A well-defined absorption peak at 695 nm, similar to that seen in the case of cytochrome c and characteristic of methionine ligation to the ferric haem, was observed. The results indicated that the haem of C357M cytochrome P450cam is possibly axially coordinated to a methionine and a histidine, analogously to cytochrome c. The circular dichroism spectra in the visible and the far-UV regions suggested that the tertiary structure of the haem cavity in the C357M mutant cytochrome P450cam was distinctly different from that in the wild-type enzyme or in cytochrome c, although the secondary structure of the mutant remained identical to that of the wild-type cytochrome P450cam. Comparison of the natures of the CD spectra in the 400 nm and 695 nm regions of the C357M mutant of cytochrome P450cam with those of horse cytochrome c suggested (R) chirality at the sulfur atom of the iron-bound methionine residue in the mutant. The redox potential of the haem centre, estimated by redox titration of the C357M mutant, was found to be +260 mV, which is much higher than that in the wild-type enzyme and similar to the redox potential of cytochrome c. This supported the concept that axial ligation of the haem plays the major role in tuning the redox potential of the haem centre in haem proteins.

The Role of Extra Fragment at the C-terminal of Cytochrome b (Residues 421–445) in the Cytochrome bc1 Complex from Rhodobacter sphaeroides

Sequence alignment of cytochrome b of the cytochrome bc1 complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with progressive deletion from this fragment (residues 421-445) were generated and characterized. The cytbDelta-(433-445) bc1 complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c1, redox potentials of cytochromes b and c1 in the cytbDelta-(427-445), cytbDelta-(425-445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc1 complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues (residues 421-432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc1 complex.

Role of the Aromatic Ring of Tyr43 in Tetraheme Cytochrome c3 from Desulfovibrio vulgaris Miyazaki F

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c 3. The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c 3. Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c 3 by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.

A multigeneration analysis of cytochrome b(562) redox variants: evolutionary strategies for modulating redox potential revealed using a library approach.

The redox potential of cytochromes sets the energy yield possible in metabolism and is also a key determinant of the rate at which redox reactions proceed. Here, the heme protein, cytochrome b(562), is used to study the in vitro evolution of redox potential within a library of variants containing the same structural archetype, the four-helix bundle. Multisite variations in the active site of cytochrome b(562) were introduced. A library of variants containing random mutations in place of R98 and R106 was created, and the redox potentials of a statistical sampling of this library were measured. This procedure was carried out for both the low- and high-potential variants of a previously studied F61X/F65X, first-generation library [Springs, S. L., Bass, S. E., and McLendon, G. L. (2000) Biochemistry 39, 6075]. The second-generation library reported here has a range of redox potentials which is greater than 40% (160 mV) of the known accessible potential among cytochromes with identical axial ligands (but different folds) and exceeds the range exhibited phylogenetically by the cytochrome c' family which internally maintains the same axial ligation and fold. A statistical analysis of the libraries examined reveals that the redox potential of WT cyt b(562) is found at the high-potential extremum of the distribution, indicating that this protein apparently evolved to differentially stabilize the reduced protein. The 2.7 A crystal structure of F61I/F65Y/R106L (low-potential variant of the second-generation library) was solved and is compared to the wild-type structure and the 2.2 A resolution structure of the F61I/F65Y variant (low-potential variant of the first-generation library). The structures indicate that charge-dipole effects are responsible for shifting the redox equilibrium toward the oxidized state in both the F61I/F65Y and F61I/F65Y/R106L variants. Specifically, a new protein dipole is introduced into the heme microenvironment as a result of the F65Y mutation, two new internal water molecules (one in hydrogen-bonding distance of Y65) are found, and in the case of F61I/F65Y/R106L (DeltaE(m) = 158 mV vs NHE), increased solvent exposure of the heme as a result of the R106L substitution is identified.

Marked hyperlactatemia associated with severe alkalemia in a patient with thrombotic thrombocytopenic purpura

This report describes a case of severe alkalemia associated with a blood lactate level greater than 13 mEq/L in a patient without evidence of hypotension or hypoxemia. The patient, who had the clinical manifestations of thrombotic thrombocytopenic purpura (TTP), developed the alkalemia from an acute respiratory alkalosis superimposed on an existing metabolic alkalosis. Profound alkalemia may impair oxygen delivery because of stronger hemoglobin-oxygen affinity, vasoconstriction, and alterations in the redox potential of cytochrome c. We suggest that the synergistic effects of a sudden, extreme alkalemia and the localized tissue hypoxia that resulted from extensive microvascular thrombi secondary to TTP caused the patient's hyperlactatemia.

Tryptophan-heme pi-electrostatic interactions in cytochrome f of oxygenic photosynthesis.

Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.

Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.

To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability.

A comparison of the redox potentials of cytochrome c3 from Desulfovibrio vulgaris Hildenborough with those from Desulfovibrio vulgaris Miyazaki F effects of amino acid substitutions on the redox potentials

Abstract The macroscopic and microscopic redox potentials of cytochrome c 3 from Desulfovibrio vulgaris Hildenborough ( Dν H) were estimated by the combined use of 1 H NMR and differential pulse polarography at two ionic strengths. The redox potentials obtained were compared with those of Desulfovibrio vulgaris Miyazaki F ( Dν MF) to see the effect of amino acid substitution in the 13 residues Although the general tendencies are similar to each other, there are distinct differences. The average redox potentials of Dν H cytochrome c 3 were lower than those of Dν MF by about 34 mV. The difference in the microscopic redox potentials was significant for hemes 1 and 3. The cooperative interaction between hemes 1 and 2 was larger in Dν H cytochrome c 3 than in Dν MF cytochrome c 3 and the former was ionic strength dependent. The microscopic redox potentials of Dν H cytochrome c 3 increased with an increase of ionic strength.

The role of heme axial histydil imidazole on the redox potentials of cytochrome [formula omitted

The role of heme axial histydil imidazole on the redox potentials of cytochrome [formula omitted