Cytochrome oxidase: pathways for electron tunneling and proton transfer

 Electrons from cytochrome c, the substrate of cytochrome oxidase, a redox-linked proton pump, are accepted by Cu_A in subunit II. From there they are transferred to the proton pumping machinery in subunit I, cytochrome a and cytochrome a ₃–Cu_B. The reduction of the latter site, which is the dioxygen reducing unit, is coupled to proton uptake. Dioxygen reduction involves a peroxide and a ferryl ion intermediate, and it is the transition between these and back to the resting oxidized enzyme that are coupled to proton pumping. The X-ray structures suggest electron–transfer pathways that can account for the observed rates provided that the reorganization energies are small. They also reveal two proton-transfer pathways, and mutagenesis experiments have shown that one is used for proton uptake during the initial reduction of cytochrome a ₃–Cu_B, whereas the other mediates transfer of the pumped protons. Received: 23 March 1998 / Accepted: 11 May 1998     

Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (azurin, cytochrome c, myoglobin) and the bacterial photosynthetic reaction center reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. The β-sheet azurin structure efficiently and isotropically mediates coupling with an exponential distance-decay constant of 1.1?Å^–1. The experimentally derived distance-decay constant of 1.4?Å^–1 for long-range ET in myoglobin and the reaction center suggests that hydrogen-bond couplings are weaker through α helices than across β sheets. The donor-acceptor interactions of systems with comparable tunneling energies fall into two coupling zones: the β zone (bounded by distance-decay constants of 0.9?and 1.15 Å^–1) includes all the β-sheet (azurin) couplings and all but one coupling in cytochrome c; the α zone (boundaries: 1.25 and 1.6?Å^–1) includes less strongly coupled donor-acceptor pairs in myoglobin and the reaction center as well as a relatively weakly coupled pair in cytochrome c.     

Redox-Bohr effect in electron/proton energy transduction: cytochrome c 3 coupled to hydrogenase works as a 'proton thruster' in Desulfovibrio vulgaris

 A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c ₃, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement. Received: 2 April 1997 / Accepted: 23 May 1997     

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction ³Zncyt + az(II) → Zncyt⁺ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrosatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.     

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^II(Cys112Asp) azurin can be reduced by excess [Ru^II(NH₃)₆]²⁺, resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c ₅₅₁ (μ?=?0.1?M sodium phosphate (pH?7.0);E°(Cu^II/I)?=?180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^II(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^II–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.     

Location of a cytochrome c binding site on the surface of flavocytochrome b 2

Saccharomyces cerevisiae flavocytochrome b ₂ couples the oxidation of L-lactate to the reduction of cytochrome c. The second-order rate constant for cytochrome c reduction by flavocytochrome b ₂ depends on the rate of complex formation and is sensitive to ionic strength. Mutations in the heme domain of flavocytochrome b ₂ (Glu63→Lys, Asp72→Lys and the double mutation Glu63→Lys:Asp72→Lys) have significant effects on the reaction with cytochrome c, implicating these residues in complex formation. This kinetic information has been used to guide molecular modelling studies, which are consistent with there being no one single best-configuration. Rather, there is a set of possible complexes in which the docking-face of cytochrome c can approach flavocytochrome b ₂ in a variety of orientations. Four cytochromes c can be accommodated on the flavocytochrome b ₂ tetramer, with each cytochrome c forming interactions with only one flavocytochrome b ₂ subunit. All the models involve residues 72 and 63 on flavocytochrome b ₂ but in addition predict that Glu237 may also be important for complex formation. These acidic residues interact with the basic residues 13, 27 and 79 on cytochrome c. Through this triangle of interactions runs a possible σ-tunnelling pathway for electron transfer. This pathway starts with the imidazole ring of His66 (a ligand to the heme-iron of flavocytochrome b ₂) and ends with the ring of Pro68, which is in van der Waals contact with the cytochrome c heme. In total, the edge-to-edge "through space" distance from the imidazole ring of His66 to the C3C pyrrole ring of cytochrome c is 13.1?Å.     

Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3

Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o3 and its transfer to CuB. The first phase occurring on a timescale of <50 ns is characterized by a volume decrease of -1.3+/-0.3 mL mol-1 and enthalpy change of 32+/-1.6 kcal mol-1. Subsequently, a volume increase of 2.9 mL mol-1 with an enthalpy change of -5.3+/-2.5 kcal mol-1 is observed with the lifetime of approximately 250 ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo3 oxidase indicating that the heme o3/CuB active site dynamics are affected by the redox state of heme b.     

Co-evolution of cytochrome c 6 and plastocyanin, mobile proteins transferring electrons from cytochrome b 6f to photosystem I

 Cytochrome c ₆ and plastocyanin are soluble metalloproteins that act as mobile carriers transferring electrons between the two membrane-embedded photosynthetic complexes cytochrome b ₆ f and photosystem I (PSI). First, an account of recent data on structural and functional features of these two membrane complexes is presented. Afterwards, attention is focused on the mobile heme and copper proteins – and, in particular, on the structural factors that allow recognition and confer molecular specificity and control the rates of electron transfer from and to the membrane complexes. The interesting question of why plastocyanin has been chosen over the ancient heme protein is discussed to place emphasis on the evolutionary aspects. In fact, cytochrome c ₆ and plastocyanin are presented herein as an excellent case study of biological evolution, which is not only convergent (two different structures but the same physiological function), but also parallel (two proteins adapting themselves to vary accordingly to each other within the same organism). Received: 4 July 1996 / Accepted: 16 September 1996     

Effects of charged peptides on electron transfer from [Fe(CN)6]4– to cytochrome c or plastocyanin

 Interactions of charged peptides, such as aspartic acid peptides (Aspptds) and lysine peptides (Lysptds), with cytochrome c (cyt c) or plastocyanin (PC) have been studied by measuring electron transfer between [Fe(CN)₆]^4– and cyt c or PC in the presence of these peptides. Aspptds, up to penta-aspartic acid, served as competitive inhibitors of electron transfer from [Fe(CN)₆]^4– to oxidized cyt c, while Lysptds, up to penta-lysine, promoted electron transfer from [Fe(CN)₆]^4– to oxidized PC. The electron transfer inhibitory effects of Aspptds are explained as competitive inhibition due to neutralization of the positively charged amino acid residues at the surface of cyt c by electrostatic interactions, whereas the electron transfer promoting effects of Lysptds may be due to formation of PC·Lysptd or Lysptd·[Fe(CN)₆]^4– complexes subsequently forming an electron transferring complex, PC·Lysptd·[Fe(CN)₆]^4–, without repulsion of the negative charges. The inhibitory effect of Aspptds and promotional effect of Lysptds became significant as the net charge or concentration of the peptides increased. The promotional effects of Lysptds decreased as the net charge of the PC negative patch was decreased by mutagenesis. Thus, charged peptides may serve as a probe for investigation of the molecular recognition character of proteins. Received: 19 May 1998 / Accepted: 27 July 1998     

Redox properties of an engineered purple Cu(A) azurin

Purple Cu(A) centers are a class of binuclear, mixed-valence copper complexes found in cytochrome c oxidase and nitrous oxide reductase. An engineered Cu(A) protein was formed by replacing a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Pseudomonas aeruginosa azurin with the corresponding portion of sequence from the Cu(A) center of cytochrome c oxidase from Paracoccus denitrificans [Proc. Natl. Acad. Sci. USA 93 (1996) 461]. Oxidation-reduction midpoint potential (E(m)) values of the Cu(A) azurin of +399+/-10 and +380+/-2mV, respectively, were determined by cyclic voltammetry and spectrochemical titration. An n value of one was obtained, indicating that the redox reaction is cycling between the mixed valence and the fully reduced states. Whereas the E(m) value of native azurin is pH dependent, the E(m) value of Cu(A) azurin is not, as expected for the Cu(A) center. Similarities and differences in the redox properties are discussed in terms of the known crystal structures of Cu(A) centers in cytochrome c oxidase and Cu(A) azurin.     

Present status and future directions of research in electron-transfer mediated by DNA

 This commentary article presents an overview of recent experimental results on DNA-mediated electron transfer (ET) from the perspective of semiclassical ET theory. The question concerning whether or not DNA can act as a wire is addressed. Much of the article focuses on a discussion of the decay of electronic coupling (β) between electron donors and acceptors with increasing donor/acceptor separation in DNA and in protein systems. In particular, the dependence of the electronic coupling itself (H _AB) on the energy gap between the tunneling energy of the reactants and the virtual ionic states of the DNA bridge is highlighted. The article concludes by suggesting that future experimental and theoretical work in this field should focus on the tunneling gap energies of the systems studied and that special attention should be paid to systems that are likely to be in the "small tunneling gap" regime. It is these systems that are expected to exhibit enhanced electronic couplings and consequently enhanced rates of long-distance ET. Received, accepted: 5 January 1998     

Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996     

Random binding of dimers to chains

We develop a probabilistic model for the binding of a small linear polymer to a larger chain. We assume that we can approximate the energy of interaction of the two chains by summing the pairwise interactions between subunits. Because the energy of interaction between a pair of subunits can depend on neighboring subunits, which we assume vary along the chain, we assign the pairwise energies of interactions according to a specified probability distribution. Thus we develop a statistical model for the binding of two molecules. While such models may not be appropriate for studying the interaction of a particular pair of molecules, they can provide insight into questions that deal with populations of molecules, such as why do MHC molecules bind peptides of a certain size? Here we analyze in detail the special case of a heterodimer binding to a polymer.     

On the mechanism of cytochrome oxidation in bacterial photosynthesis Quantum tunnelling effects revisited

We propose that the unique temperature dependence of the Chance-De Vault cytochrome oxidation reaction in Chromatium is not due to a transition from low-temperature nuclear tunnelling to a high-temperature activated electron transfer (ET), but rather originates from two parallel ET processes from two distinct low-potential cytochromes to the bacteriochlorophyll dimer cation. These involve a slow activationless process, which dominates at low temperatures (T 120 K) and an activated process, which is practically exclusive at high temperatures. This conjecture provides plausible nuclear and electronic coupling terms and structural data for the two cytochrome oxidation reactions.     

Crystal structure of cytochrome c' from Rhodocyclus gelatinosus and comparison with other cytochromes c'

Cytochromes c' are heme proteins found in photosynthetic and denitrifying bacteria, where they are presumably involved in electron transport. The cytochrome c' isolated from the bacterium Rhodocyclus gelatinosus (RGCP) forms a homodimer with each polypeptide containing 129 residues. It has been crystallised in ammonium sulfate at pH?6. Crystals belong to space group P3₁21 with cell parameters a?=?70.2?Å and c?=?126.8?Å, which corresponds to a dimer in the asymmetric unit (VM?=?3.5?ų?/?Da). The crystal structure of RGCP was solved by the molecular replacement method and refined using data to 2.5-Å resolution. The final crystallographic R factor was 17.9% for all reflections (above 2?σ) in the resolution range 27.4 to 2.5?Å. The refined model includes 1876 non-hydrogen protein atoms and 56 water molecules. As typical of c–type cytochromes, the heme group is covalently bound to Cys-X-Y-Cys-His through thio-ether bonds, and His123 occupies the fifth axial coordination position. On the vacant "distal" site, Phe16 blocks the direct access to the sixth coordination site, which is in a predominantly hydrophobic environment. In spite of the low sequence homology among cytochromes c' the overall fold is similar. The monomer structure consists of 4 anti-parallel α-helices and has random coils in the loops between the helices, and at the N- and C-termini. The subunits cross each other to form an X shape.     

The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example

 The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only. Received: 11 July 1995 / Accepted: 30 October 1995     

The kinetics of electron entry in cytochromec oxidase

Summary The kinetics of electron entry in beef heart cytochromec oxidase have been studied by stopped-flow spectroscopy following chemical modification of the Cu_A site with mercurials. In this derivative Cu_A is no longer reducible by cytochrome c while cytochromea may accept electrons from the latter with rates comparable to the native enzyme. The results indicate that Cu_A is not the exclusive electron entry site in cytochromec oxidase.     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999