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Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain that catalyzes respiratory reduction of dioxygen ([O.sub.2]) to water in all eukaryotes and many aerobic bacteria. CcO, and its homologs among the heme-copper oxidases, has an active site composed of an oxygen-binding heme and a copper center in the vicinity, plus another heme group that donates electrons to this site. In most oxidoreduction enzymes, electron transfer (eT) takes place by quantum-mechanical electron tunneling. Here we show by independent molecular dynamics and quantum-chemical methods that the heme-heme eT in CcO differs from the majority of cases in having an exceptionally low reorganization energy. We show that the rate of interheme eT in CcO may nevertheless be predicted by the Moser-Dutton equation if reinterpreted as the average of the eT rates between all individual atoms of the donor and acceptor weighed by the respective packing densities between them. We argue that this modification may be necessary at short donor/ acceptor distances comparable to the donor/acceptor radii. biological electron transfer | heme-copper oxidases | Moser-Dutton ruler | reorganization energy doi/ 10.1073/pnas.1005889107

Bacterial cytochrome c maturation occurs at the outside of the cytoplasmic membrane, requires transport of haem b across the membrane, and depends on membrane-bound cytochrome c haem lyase (CCHL), an enzyme that catalyses covalent attachment of haem b to apocytochrome c. Epsilonproteobacteria such as Wolinella succinogenes use the cytochrome c biogenesis system II and contain unusually large CCHL proteins of about 900 amino acid residues that appear to be fusions of the CcsB and CcsA proteins found in other bacteria. CcsBA-type CCHLs have been proposed to act as haem transporters that contain two haem b coordination sites located at different sides of the membrane and formed by histidine pairs. W. succinogenes cells contain three CcsBA-type CCHL isoenzymes (Nrfl, CcsA1 and CcsA2) that are known to differ in their specificity for apocytochromes and apparently recognize different haem c binding motifs such as [CX.sub.2]CH (by CcsA2), [CX.sub.2]CK (by Nrfl) and [CX.sub.15]CH (by CcsA1). In this study, conserved histidine residues were individually replaced by alanine in each of the W. succinogenes CCHLs. Characterization of Nrfl and CcsA1 variants in W. succinogenes demonstrated that a set of four histidines is essential for maturing the dedicated multihaem cytochromes c NrfA and MccA, respectively. The function of W. succinogenes CcsA2 variants produced in Escherichia coli was also found to depend on each of these four conserved histidine residues. The presence of imidazole in the growth medium of both W. succinogenes and E. coli rescued the cytochrome c biogenesis activity of most histidine variants, albeit to different extents, thereby implying the presence of two functionally distinct histidine pairs in each CCHL. The data support a model in which two conserved haem b binding sites are involved in haem transport catalysed by CcsBA-type CCHLs. DOI 10.1099/mic.0.042838-0

Cytochrome c (Cyt c) was covalently immobilized on a boron-doped nanocrystalline diamond (BDND) electrode via surface functionalization with undecylenic acid methyl ester and subsequent removal of the protecting ester groups to produce a carboxyl-terminated surface. Cyt c-modified BDND electrode exhibited a pair of quasi-reversible and well-defined redox peaks with a formal potential ([E.sup.0]) of 0.061V (vs Ag/AgCl) in 0.1 M phosphate buffer solution (pH 7.0) and a surface-controlled process with a high electron transfer constant ([k.sub.s]) of 5.2 [+ or -] 0.6 [s.sup.-1]. The electrochemical properties of as-deposited and Cyt c-modified boron-doped microcrystalline diamond (BDMD) electrodes were also studied for comparison. Investigation of the electrocatalytic activity of the Cyt c-modified BDND electrode toward hydrogen peroxide ([H.sub.2][O.sub.2]) revealed a rapid amperometric response (5 s). The linear range of response to [H.sub.2][O.sub.2] concentration was from 1 to 450 [micro]M, and the detection limit was 0.7 [micro]M at a signal-to-noise ratio of 3. The stability of the Cyt c-modified BDND electrode, in comparison with that of the BDMD and glassy carbon counterpart electrodes, was also evaluated.

A time-dependent perturbation theory was used for examining the vibrational energy relaxation (VER) of amide I modes in cytochrome c solvated with water. Findings reveal the relaxation of the amide I modes in protein with subpicosecond time scales.

The analysis describes the generation of singlet oxygen from the decomposition of polyunsaturated lipid peroxide, [alpha]-linolenic acid hydroperoxide (LAOOH) by employing various heme-proteins such as cytochrome c and lactoperoxidase. The various results obtained have proved to be extremely helpful for understanding the damage mechanism of a cell membrane by singlet oxygen and the different radicals generated in the peroxidation and decomposition of lipids.

A kinetic model is provided for the time constant for interconversion of disparate unfolded chain conformations of a small globular protein, cytochrome c, in the presence of guanidine hydrochloride denaturant. The kinetic model fits both the reaction rate constants and the spectra of the intermediates to obtain a quantitative value for the conformational diffusion time constant.

An all-atom large-scale steered molecular dynamics (SMD) simulation is combined with evolutionary studies and binding free energy analysis in order to propose and explore dissociation pathways of cytochrome (cyt [c.sub.2]) from the reaction center (RC) of Rhodobacter sphaeroides embedded in a membrane without the light-harvesting complex I. The results have shown that the redox-dependent variations might lead to changes in structural flexibility, behavior of the interfacial water molecules and in the binding free energy of the complex.

The effect of the polarity and viscosity of the solvent medium on the unfolding and refolding reactions of Z[n.sup.II]-substituted cytochrome c at room temperature is examined. The results suggest an important role for the solvent in the kinetic control of protein-folding trajectories on the energy landscape.

This article reports on the extraction behavior of heme proteins from an aqueous phase into ionic liquids (ILs) with dicyclohexano-18-crown-6 (DCH18C6), and the structure-function relationship of cytochrome c (Cyt-c) dissolved in ILs. We have found that DCH18C6 enables transfer of Lys-rich proteins into ILs via supramolecular complexation. The hydrophobicity and functional groups of ILs have a great influence on protein partitioning, and a hydroxyl group-containing IL with DCH18C6 is capable of the quantitative partitioning of Cyt-c. On the other hand, protein transfer using conventional organic solvents is negligibly small. IN-visible, CD, and resonance Raman spectroscopic characterizations indicate that the sixth ligand Met 80 in the heme group of the Cyt-c-DCH18C6 complex in IL is replaced by other amino acid residues of the peptide chain and that a non-natural, six-coordinate, low-spin ferric heme structure is induced in IL. Solubilization of Cyt-c in IL causes the environmental change of the heme vicinity of Cyt-c, which triggers the functional conversion of Cyt-c from an electron-transfer protein to peroxidase. The Cyt-c-DCH 18C6 complex in IL provides remarkably high peroxidase activity compared with native Cyt-c, because of enhancement of the affinity for [H.sub.2][O.sub.2].

The effects of porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively.

We have proposed novel surface-imprinted beads for selective separation of cytochrome c (cyt c) by N-methacryloyl-(L)-histidine--copper(II) [MAH--Cu(II)] as a new metal-chelating monomer via metal coordination interactions and histidine template. We have combined molecular imprinting with the ability of histidine to chelate metal ions to create ligand exchange beads suitable for the binding of cyt c (surface histidine exposed protein). The histidine imprinted beads were produced by suspension polymerization of MAH--Cu(II)--L-histidine and ethylene glycol dimethacrylate. After polymerization, the template (L-histidine) was removed from the beads using methanolic KOH, thus getting histidine imprinted metal--chelate beads. L-Histidine imprinted metal--chelate beads can be used several times without considerable loss of cyt c adsorption capacity. The association constant ([K.sub.a]) for the specific interaction between the template imprinted polymer and the template (L-histidine) itself were determined by Scatchard plots using L-histidine imprinted beads and found as 58 300 [M.sup.-1]. Finally, we have used these histidine imprinted beads for cyt c and ribonuclease A (surface histidine exposed proteins) and enantiometric separation of D- and L-histidine by FPLC.

An extensively misfolded kinetic intermediate is described in the folding reaction of the horse alkaline ferrocytochrome c, which further requires a large-scale unfolding to fold correctly to the native state. The analysis reveals that the failure of the polypeptide chain to achieve the transition state via stochastic search leads to the high level of observed misfolding, as the rate of unfolding of the misfolded intermediate also limits the rate of protein folding.

Several studies and experiments are conducted to show that the major proton-transfer pathway in the buried site of cytochrome c oxidase (CcO) is the D-pathway that begins in the absence of subunit III on the inner protein surface. The results demonstrate that the subunit III makes a barrier for the protons and hence prevents them from directly accessing the internal depths of the D-pathway, though it also helps in increasing the rate at which the protons are transferred in the pathway.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.05.026 Byline: Dhyan Chandra (1), Shawn B. Bratton (2), Maria D. Person (2), Yanan Tian (3), Angel G. Martin (4), Mary Ayres (5), Howard O. Fearnhead (4), Varsha Gandhi (5), Dean G. Tang (1)(6) Abstract: Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death. Author Affiliation: (1) Department of Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957, USA (2) Division of Pharmacology/Toxicology, College of Pharmacy, University of Texas at Austin, Austin, TX 78712, USA (3) Department of Veterinary Physiology and Pharmacology, MS 4466, Texas A&M University, College Station, TX 77843, USA (4) Apoptosis Section, Laboratory of Protein Dynamics and Signaling, NCI-Frederick, Frederick, MD 21702, USA (5) Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA (6) Program in Molecular Carcinogenesis of Graduate School for Biomedical Science, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA Article History: Received 22 June 2005; Revised 6 January 2006; Accepted 3 May 2006 Article Note: (miscellaneous) Published: June 29, 2006

A nonspectroscopic chemical method is employed to determine the binding site on horse cytochrome involved in its adsorptive immobilization on a COOH-terminated self-assembled monolayer (SAM). The factors that can complicate or distort data interpretation and the generality of differential modification relative to existing in situ methods for protein orientation determination are discussed.

The structure-function studies on the protein were carried out and its interaction with sphaeroides heme protein (SHP) was examined to gain further insight into the properties and role of diheme cytochrome c (DHC). Evidence of complementary binding, favorable midpoint potentials, and effect electron transfer in the DHC-SHP system is provided.

A study was conducted about the redox behavior of native and mutated cytochrome c vectorially immobilized on unmodified gold electrodes at varying temperatures and pH values. The immobilized low-pH conformers of both native and the C102T/N62C variant of yeast iso-1-cytochrome c are found to be electroactive and to reversibly convert to the 'neutral' His,-Met-ligated form with pH.

The five-coordinate NO-bound heme in cytochrome c' from an overexpressing variant of denitrifying R. sphaeroides 2.4.3 was investigated by proton, nitrogen, and deuterium Q-band ENDOR (electron nuclear double resonance). ENDOR frequencies from heme meso-protons, assigned with reference to porphyrin models, were determined to result from an anisotropic hyperfine tensor.

The activation of peroxidase activity of cytochrome (cyt c) by cardiolipin (CL) and hydrogen peroxide is characterized quantitatively. Findings suggest that electrostatic CL/ cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost.

Cytochrome c(sub1) of Rhodobacter spaeroides ubiquinol-cytochrome c oxidoreductase contains several insertions and deletions that distinguish it from the complex of other higher organisms. Cleavage of cytochrome c(sub 1) between the two cysteines followed by gel electrophoresis shows two fragments only under reducing conditions confirming the existence of disulfide bridge which is essential in maintaining the structural integrity of cytochrome c(sub 1).

Purified bovine heart cytochrome c oxidase (CcO) was extracted from aqueous solution into hexane in the presence of phospholipids and calcium ions. The enhanced rate of binding of cyanide to heme a(sub 3) was described by the destabilization of an intrinsic ligand, located at the catalytic site, that was triggered by the reduction of heme a and Cu(sub A).

A desorption electrospray ionization (DESI) source is coupled to an ion mobility time-of-flight mass spectrometer for the analysis of protein structures. The results have indicated that the DESI experiment is gentler than ESI and under appropriate conditions it is possible to preserve structural information throughout the DESI process.

A study demonstrates that D2O substitution can have large effects on the association modes and electron transfer (ET) reactivity of yeast iso-1-cytochrome c (yCc) with either cytochrome c peroxidase (CcP) in crystals or surface-functionalized electrodes. Electrochemistry confirms that solvent exchange produces minimal changes to potential and activation energies of yCc on solvent exchange, and reveals two isotope effects that occur with different time constants.

The applicability of Badger's rule to heme and non-heme iron-oxygen bonds is examined to gain insights into the protonation state of enzymatic ferryl species. The long Fe-O bonds reported in the crystal structures of the ferryl forms of myoglobin, cytochrome c peroxidase and catalase deviate substantially from the values predicted by Badger's rule, while the short Fe-O bonds obtained from X-ray absorption measurements are in good agreement with Badger's rule.

Cytochromes c, are among the most studied proteins, possibly due to their relatively high thermodynamic stability and their red color, which makes protein purification easier and the three-dimensional structure of mitochondrial cytochrome c has been solved in the 1970s.The small size, high solubility, high helical content and the presence of the heme cofactor have allowed mitochondrial and some bacterial cytochromes c to be studied through spectroscopic techniques, which have contributed to making cytochrome c a very popular protein among biochemist and biophysicists.

The redox-active centers of cytochrome c oxidase from P. denitrificans together with key amino acids, W164, E278, R473 and R474 are discussed. The DELTA-propionate of heme a(sub 3) is stabilized by hydrogen bonds from W164 and R473 and by charge interactions with R473 and R474.

Density functional theory calculations are performed on the active species of cytochrome c peroxidase (CcP) and ascorbate paroxidase (APX) models. The calculations show that small external perturbations are sufficient to change the electronic state of the active species and subsequently its catalytic properties.

The alkaline transition kinetics of a Lys 73 ->His (H73) variant of iso-1-cytochrome c are triggered by three ionizable groups is presented. It shows that the Ala 79 mutation causes formation of the alkaline conformer to depend on [NaCl], the dependence saturates at 500 mM NaCl, and the thermodynamics of alkaline state formations for A79H73 and H73 variants become identical.

A study about internal electron transfer (ET) to heme a(sub3) during anaerobic reduction of oxidized bohive heart cytochrome c oxidase (CcO) is presented. The findings shows that the rate of ET to heme a(sub 3) in the as-isolated resting enzyme and in CcO 'activated' by reaction of fully reduced enzyme with O(sub 2) is the same.

A methodical order was developed whereby the influences of surface area, pore size, extent of order, particle size, surface potentials, isoelectric points, pH, and ionic strength on immobilization were explored. A systematic methodology for the adsorption of proteins onto mesoporous silicates is described.

An aspartic acid is introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects are examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity.

The time course and site(s) of tyrosine nitration in horse cytochrome c by peroxynitrite is analyzed and a method of purifying nitrated cytochrome c product by cation-exchange high performance liquid chromatography (HPLC) is developed. The use of purified and well-characterized mono- and dinitrated cytochrome c species highlights the influence of nitration of specific tyrosines in cytochrome c functions.

An investigation on the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution is reported. Horse heart cytochrome c (cyt c) is found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea.\

Optical spectroscopy, MCD and stopped-flow for the pH sensitivity of spectral features are used to investigate the three forms of cytochrome c oxidase, fully oxidized CcO (CcO-O), oxidized CcO complexed with cyanide (CcO.CN), and mixed valence CcO, in which both heme a3 and Cu(sub B) are reduced and stabilized by carbon monoxide (MV.CO). Glu60 of subunit II, located at the entrance of the proton conducting K-channel, is the proteolytic residue that interacts with both hemes through a hydrogen-bonding network.

Molecular dynamics (MD) simulations of the reaction center (RC)cyt c2 encounter complex for both redox states of cyt c2 are reported. The results revealed that single-file water molecules serve as a redox switch where water molecules could sense the change in electrostatic potential leading to facilitated undocking after oxidation.

Detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths (~100 mM), two binding sites were found for the reduced wildtype cytochrome (K(sub D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5 fold).

A class of polyanionic copper porphyrin dimers is shown to selectively increase the susceptibility of cytochrome c to proteolysis through binding-inducted disruption of tertiary and secondary structure. The findings show that the proteolytic acceleration is catalytic in nature, requiring only a fraction of an equivalent of metalloporphyrin to effect complete, rapid digestion in the presence of protease.

The pH dependence of the kinetics of the conformational change between the native state and His 73-ligated alkaline state of a His 73 variant of iso-1-cytochrome c demonstrates that three ionizable group affect this process. The results are discussed in terms of the impact of ionization equilibria on protein folding mechanisms.

Heme proteins undergo vibrational energy relaxation (VER) following ligand binding or dissociation.Classical molecular dynamics simulation is used to observe the process of heme cooling in aqueously solvated cytochrome c following the direct excitation of the porphyrin chromophore.

Conventional electrochemical techniques are applied to the electron transfer of cytochrome c protein immobilized on the surface of SAM modified electrodes. Chemical control of adsorption allows the accurate determination of heterogeneous unimolecular rate constants for the electron exchange between the SAM modified metal electrode and the cytochrome c.

Most of the native cytochrome c conformations are maintained at high pH. The alkaline form of cyt c possess higher flexibility than that of the native protein, as indicated by heteronuclear NOE data,

The kinetic experiments, calculations of electrostatic properties and a new theoretical treatment of pH effects are combined together to study the effects of pH on protein association. The anomalous pK(sub a) values of certain amino acid residues in both cytochrone c and plastocyanin are seen to be significant for their biological functions.

Low-temperature visible absorption spectral analysis for recombinant Thermus thermophilus cytochrome c552 is reported. Quantum chemical calculations are performed to determine quantitatively the effect of heme conformation, axial ligand, peripheral substituents and local electric fields on the electronic spectra.

Cytochrome c (cyt c) is a member of the ubiquitous class of heme proteins, which function as reversible redox carriers in electron-transport chains. Ligand photolysis and subsequent electronic and structural relaxation, followed by ligand recombination, in ferrous cyt c is studied using ultrafast laser spectroscopy.

The coupling of protonation and reduction is crucial in many biological charge transfer reactions and is known as redox Bohr effect and it is caused by electrostatic interactions between protonatable and redox-active groups. The study discusses the energetic of protonation and redox reactions throughout the whole investigated pH and solution redox potential range.

An excess of the [A-Ru(bpy)2(H2O)]-His33-cyt c diastereoisomer is formed from the reaction of rac-[Ru(bpy)2(H2O)(sub 2)](super 2+) (bpy = 2,2' bipyridine) with horse heart cytochrome c. Intermolecular electron-transfer rates were studied by oxidative pulse radiolysis and the results agree with flash photolysis measurements.

Highly anisotropic electronic coupling is revealed in the electrochemistry of surface-modified cytochrome c that is bound electrostatically to alkanethiol self-assembled monolayers (SAMs). It is shown that lysine -13 is directly involved in coupling the protein to the SAM carboxylate terminus.

The potential-controlled electron-transfer reaction between cyrochrome c and 1, 1'-dimethylferrocene at a liquid-liquid interface is reported. In this approach to protein electrochemistry, the electron transfer process is apparently transport controlled, rather than adsorption limited.

Hydrophobic electron donors and acceptors are encapsulated within the hemicarcerand and photoinduced electron transfer (ET) between theZn-substituted cytochrome c and the host-guest complexes is used for probing the association between the negatively charged hemicarceplex and the positively charged protein. The strong association with the protein is attributed to the flexible conformation and adaptable charge distribution of the hemicarcerand, which has allowed for surface-matching with the cytochrome.

Several NMR studies are conducted to explain the numerous redox linked conformational changes that take place in cytochrome [C.sub.3] obtained from Desulfovibrio desulfuricans ATCC 27774. The structural rearrangements taking place in the compound are shown to be highly dependent on the pH.