Electron transfer between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa

The electron transfer equilibrium and kinetics between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa have been studied. The equilibrium constant K = ([Cyt(III)] . [Az(I)])/([Cyt(II)] . [Az(II))]) = 0.5 at 25 degrees C is about seven times smaller than that observed between the cytochrome c551 and the titrations confirmed a 43-mV difference between the mid-point potentials of +266 mV and +309 mV for the Alcaligenes and Pseudomonas azurins respectively. The kinetics of the reaction between Alcaligenes azurin and Pseudomonas cytochrome c551 were investigated by the temperature-jump chemical relaxation method. Only a single relaxation mode was observed throughout the range of concentrations and temperatures examined. Thus, the slow relaxation time observed in the reaction between P. aeruginosa azurin and cytochrome c551 is not observed with the Alcaligenes azurin. The simplest mechanism that can therefore be ascribed to the investigated system is: [formula: see text]. This scheme is similar to that proposed earlier for the reaction between P. aeruginosa azurin and cytochrome c551 but does not involve the conformational transition proposed for azurin. The specific rates for the electron transfer are still fast: 1.8 x 10(6) M-1 . s-1 and 3.0 x 10(6) M-1 . s-1 respectively at 25 degrees C.     

Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase

The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.     

Resolution of two distinct electron transfer sites on azurin

Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled upon reduction by Cr(II)aq ions, yielding a substitution-inert Cr(III) adduct on the protein surface. We investigated the effect of this chemical modification on the reactivity of azurin with two of its presumed partners in the redox system of the bacterium. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(III)-labeled azurin by O2 was found to be unaffected by the modification. The kinetics of the electron exchange reaction between native or Cr(III)-labeled azurin and cytochrome c551 were studied by the temperature-jump method. Though similar chemical relaxation spectra were observed for native and modified systems, they differ quantitatively. Analysis of the concentration dependences of the relaxation times and amplitudes showed that both obey the same mechanism but that the specific reaction rates of the Cr(III)-modified protein are attenuated. This decreased reactivity of Cr(III)-labeled azurin toward one of its physiological partners suggests the involvement of the labeled region in the electron transfer reaction with cytochrome c551. Furthermore, the presence of a second active site, involved in the reduction of cytochrome oxidase, is suggested by the results.     

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.     

Kinetics of the generation of a photo-induced electric potential in chromatophores of photosynthetizing bacteria

Flash-induced formation of an electric potential difference (delta psi) was monitored by a direct method in chromatophores associated with the collodion phospholipid membrane. In Rhodospirillum rubrum and Rhodopseudomonas sphaeriodes chromatophores, the kinetics of delta psi generation exhibit fast (tau less than or equal to 0.3 microseconds) and slow (tau congruent to 200 microseconds) phases, the latter observed in the presence of exogenous quinones. Comparison of the kinetic and potentiometric characteristics of the process with those of electron transport reactions suggests that the fast phase of delta psi rise is due to charge separation between the primary electron donor, P870, and primary electron acceptor QIFe; the slow phase, which is inhibited by o-phenanthroline, is due to electron donation from QIFe to the secondary acceptor, quinone QII. The kinetics of delta psi decay include components arising form the recombination of primary separated charges (tau congruent to 30 ms) and from the passive discharge of the membrane (tau congruent to 400 ms; tau congruent to 1400 ms). From a redox titration of the photo-induced electric signal and the photo-induced absorption changes of P870 at different pH meanings, the value of pK for the primary acceptor FeQI was found to be 7.4 in Rps. sphaeroides chromatophores. In Chromatium minutissimum, a phase ( tau congruent to 20 microseconds) was observed in addition to those seen in Rps. sphaeroids and R. rubrum which was explained by the reduction of P890+ from the high potential cytochrome c555. Possible distribution of the electron transport components in the chromatophore membrane are discussed.     

Viscosity dependence of ethidium-DNA intercalation kinetics

The kinetics of ethidium intercalation into double-stranded poly[d(G-C)] were investigated by use of repetitive pressure-jump chemical relaxation at 20 degrees C in low ionic strength (0.1 M NaCl) aqueous buffers containing either glycerol or methanol. The viscosity of the various solvents differed by more than an order of magnitude while other physical properties (e.g., dielectric constant) remained approximately constant. The single-reciprocal kinetic relaxation time (tau -1) increases linearly with DNA concentration. The observed association rate constant is lower in all organic-aqueous mixtures than in water and is inversely proportional to the viscosity. These results provide evidence for an additional step in the intercalation mechanism which is identified as an obligatory DNA conformational change preceding ethidium intercalation. From the data presented, the equilibrium constant of this local conformational change is approximately 10(-3), i.e., greatly favoring the structure incapable of intercalation. The corresponding kinetics were not directly determined; however, in order to be consistent with all of the data the forward and/or reverse rate constants of the conformational change must be larger than the rate of the intercalation reaction. Thus, it is proposed that the rate of the conformational change back to the nonintercalating B-DNA structure is greater than approximately 500 s-1, implying a rate of opening greater than approximately 0.5 s-1, in agreement with other hydrogen exchange and NMR data. The observed overall rate constant for the dissociation of ethidium is inversely proportional to the solvent density, possibly reflecting a dependence on the solvent free volume. The overall volume change of intercalation is less negative in the organic-aqueous solvent mixtures than in water.     

pH dependence of the reduction-oxidation reaction of azurin with cytochrome c-551: role of histidine-35 of azurin in electron transfer

A fluorescence quenching experiment confirms that in the redox reaction between cytochrome c-551 and azurin, protein complexing is negligible. Azurin-pH indicator T-jump experiments show that Pseudomonas aeruginosa (Ps.) azurin exhibits a slow time constant, tau, in its return to pH equilibrium but Alcaligenes faecalis (Alc.) azurin does not. The decrease of l/tau with increasing pH shows that the rate-determining process is a slow transformation of the imidazolium form of histidine-35 from a conformation where it cannot ionize to one in which it can. The fast relaxation time constant of the redox reaction varies little with pH, but the slow time constant increased by a factor of approximately 2.5 increasing pH between pH 5 and pH 8. The corresponding amplitudes, especially the slow one, vary with pH. On the basis of all the present evidence it is concluded that, while some differences of redox reactivity do occur on protonation, these differences are not major. In general, the two proteins cyt c-551 and azurin react with each other with rates only weakly dependent upon pH. A classical pH titration was carried out on the reduced and oxidized form of Ps. and Alc. azurin with the result that two protons were released between pH 6 and pH 8, in the former from His-35 and -83 and in the latter from His-83 and Ala-1.     

The electron transfer system of Pseudomonas aeruginosa: a study of the pH-dependent transitions between redox forms of azurin and cytochrome c551

Fast reaction kinetic experiments on the electron transfer reaction between azurin and cytochrome c₅₅₁ isolated from Pseudomonas aeruginosa confirmed the existence of two redox forms of reduced azurin previously reported. The pH dependence of the amplitudes of the relaxation processes observed in temperature jump experiments indicate that these two redox forms are in pH dependent equilibrium. The pH independence of the overall equilibrium constant indicates that redox active and inactive forms of cytochrome c₅₅₁ may also exist. Evidence that reduced cytochrome c₅₅₁ undergoes a pH transition is given by optical spectrophotometry. The nature of the transition is discussed in the context of recent nmr studies and in terms of the Marcus theory of electron transfer. The metabolic consequences of these transitions are also discussed.     

Energy-generating enzymes of Burkholderia cepacia and their interactions with macrophages

We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of alpha(2)-macroglobulin. Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues of Pseudomonas aeruginosa azurin and cytochrome c(551), which are normally involved in electron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium of B. cepacia strain 71 enriched with the azurin and cytochrome c(551) homologues triggered apoptosis in macrophages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochrome c(551) antibodies greatly reduced cell death. We cloned and hyperexpressed a gene from B. cepacia strain 71 that encodes the homologue of P. aeruginosa azurin. Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars. The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages. This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.     

1H NMR investigation of cytochrome cd1: complexes with electron-donor proteins

Mixtures of the dissimilatory nitrite reductase cytochrome cd1 from Pseudomonas aeruginosa and potential electron-donating proteins were prepared in both fully oxidized and fully reduced states and examined by 1H NMR spectroscopy. The relatively narrower lines of the donor proteins enabled them to be clearly observed in spectra in the presence of significant amounts of the high molecular weight cd1. Mixtures of the physiological donor (Pseudomonas ferrocytochrome c-551) and ferrocytochrome cd1 showed specific line-broadening effects on the resonances of c-551 that depended on the mole ratio of c-551 to cd1. The experimental broadening fit a model in which c-551 is in intermediate or fast exchange between free solution and a complex with cd1, with an association constant for the complex in excess of 10(4) M-1. The model yields a minimum estimate for the forward bimolecular rate constant of 5 X 10(7) M-1 s-1 and suggests that the actual value may be much larger. The complexation was independent of pH in the range of 6-8, was independent of ionic strength over a salt concentration range of 20-1000 mM, and possessed a low thermal activation barrier. Mixtures of ferricytochrome c-551 and ferricytochrome cd1 showed no observable NMR perturbations, indicating that any hypothetical complex involving the oxidized forms must follow different dynamical and/or equilibrium conditions. No observable NMR perturbations existed in spectra of mixtures of cd1 and mammalian cytochrome c or Pseudomonas azurin in either oxidation state.     

Pseudomonas aeruginosa cytochrome oxidase. Product inhibition by low thermodynamic driving force

The steady-state kinetics of Pseudomonas aeruginosa cytochrome oxidase were studied. Reduced cytochrome c551 and azurin from the same bacteria were used as the electron-donating substrates, while dioxygen served as the electron acceptor. Oxidized cytochrome c551 and azurin exhibited product inhibition of the reaction. However, apo-azurin and azurin derivatives in which the copper was substituted by the redox-inert ions Ni2+, Co2+, Cd2+ and Zn2+, did not show any effect on the kinetics. These observations implied that complex formation between the substrates or the products and the enzyme is not a rate-limiting step and is not the cause for product inhibition. The integrated rate law for a reaction scheme in which we assumed that complex formation was not rate limiting was fitted to the complete reaction traces. The results suggested that it is the low thermodynamic driving force, expressed in the small differences in redox potential between the substrates and heme c of the enzyme, which cause the observed product inhibition.     

Electron transfer between azurin and cytochrone c-551 from Pseudomonas aeruginosa.

The electron-transfer reaction between azurin and cytochrome c1 isolated from Pseudomonas aeruginosa was investigated by rapid-reaction techniques. Temperture-jump studies clearly reveal two chemical relaxations, the amplitudes of which have ikentical spectral distributions, but relaxation times show different dependencies on reactant concentrations. Stopped experiments also showed complex kinetics. A model is proposed which is consistent with the kinetic and equilibrium data obtained. The central feature of this model is the proposal that two intercenvertible forms of reduced azurin exist in solution, only one of which si able to participate directly in the electron-transfer reaction with cytochrome c-551. Support for the hypothesis that two forms of reduced azurin exist is derived from studies on the electron-transfer reaction between azurin and Pseudomonas cytochrome oxidase. The possible physiological significance of such a situation is discussed.     

X-ray structure of methanol dehydrogenase from<Emphasis Type="Italic"> Paracoccus denitrificans</Emphasis> and molecular modeling of its interactions with cytochrome<Emphasis Type="Italic"> c</Emphasis>-551i

The X-ray structure of methanol dehydrogenase (MEDH) from Paracoccus denitrificans (MEDH-PD) was determined at 2.5 A resolution using molecular replacement based on the structure of MEDH from Methylophilus methylotrophus W3A1 (MEDH-WA). The overall structures from the two bacteria are similar to each other except that the former has a longer C-terminal tail in each subunit and shows local differences in several insertion regions. The "X-ray sequence" of the segment alphaGly444-alphaLeu452 was established, including one insertion and seven replacements compared with the reported sequence. The primary electron acceptor of MEDH-PD is cytochrome c-551i (Cyt c551i). Based on the crystal structure of MEDH-PD and of the published structure of Cyt c551i, their interactions were investigated by molecular modeling. As a guide and starting point, the covalently attached cytochrome and PQQ domains of the alcohol dehydrogenase from Pseudomonas putida HK5 (ADH2B) were used. In the modeling, two molecules of Cyt c551i could be accommodated in their interaction with the MEDH heterotetramer in accordance with the two-fold molecular symmetry of the latter. Two models are proposed, in both of which electrostatic and hydrogen bonding interactions make major contributions to inter-protein binding. One of these models involves salt bridges from alphaArg99 of MEDH to the heme propionic acids of Cyt c551i and the other involves salt bridges from alphaArg426 of MEDH to Glu112 of Cyt c551i. Both involve salt bridges from alphaLys93 of MEDH to Asp75 of Cyt c551i. The size and nature of the cytochrome/quinoprotein heterodimer interfaces and calculations of electronic coupling and electron transfer rates favor one of these models over the other.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Modification of the electron-transfer sites of Pseudomonas aeruginosa azurin by site-directed mutagenesis

Site-directed mutagenesis of the structural gene for azurin from Pseudomonas aeruginosa has been used to prepare azurins in which amino acid residues in two separate electron-transfer sites have been changed: His-35-Lys and Glu-91-Gln at one site and Phe-114-Ala at the other. The charge-transfer band and the EPR spectrum are the same as in the wild-type protein in the first two mutants, whereas in the Phe-114-Ala azurin, the optical band is shifted downwards by 7 nm and the copper hyperfine splitting is decreased by 4.10(-4)/cm. This protein also shows an increase of 20-40 mV in the reduction potential compared to the other azurins. The potentials of all four azurins decrease with increasing pH in phosphate but not in zwitterionic buffers with high ionic strength. The rate constant for electron exchange with cytochrome c551 is unchanged compared to the wild-type protein in the Phe-114-Ala azurin, but is increased in the other two mutant proteins. The results suggest that Glu-91 is not important for the interaction with cytochrome c551 and that His-35 plays no critical role in the electron transfer to the copper site.     

Intramolecular electron transfer in cytochrome c oxidase: a cascade of equilibria.

Intramolecular electron redistribution in cytochrome c oxidase after photolysis of the partially reduced CO-bound enzyme was followed at a number of different wavelengths by absorption spectroscopy. Spectra were constructed for the first two phases of this process. The first phase (tau = 3 microseconds) has a spectrum essentially identical to the difference between the Fea and Fea3 reduced-minus-oxidized spectra, indicating a 1:1 stoichiometry between the amount of Fea3 oxidized and Fea reduced. It is not necessary to invoke reduction or oxidation of other redox carriers in this phase. The second phase (tau = 35 microseconds) spectrum appears to be a linear combination of the Fea3 and Fea reduced-minus-oxidized difference spectra, reflecting the oxidation of four parts of Fea3 for every part of Fea oxidized. This process can be described in terms of transfer to CuA of electrons from the Fea3<==>Fea equilibrium system established in the first phase. The relative contributions of Fea3 and Fea in the second phase allow us to calculate the equilibrium constant for Fea3<==>Fea electron exchange, which yields a delta Em of 36 mV for the two centers (Fea3 more positive). Together with the apparent rate constant for the fast phase, this equilibrium constant yields, in turn, the forward (kf) and reverse (kr) rates for electron transfer from Fea to Fea3 as follows: kf = 2.4 x 10(5) s-1 and kr = 6 x 10(4) s-1. kf is much faster than any observed step in the reaction of the reduced enzyme with O2. Thus, the catalytic mechanism of O2 reduction to water is not rate-limited by electron transfer from Fea to the binuclear Fea3/Cu(B) site.     

Ligand binding and the catalytic reaction of cytochrome caa(3) from the thermophilic bacterium Rhodothermus marinus

The ligand-binding dynamics and the reaction with O(2) of the fully (five-electron) reduced cytochrome caa(3) from the thermohalophilic bacterium Rhodothermus (R.) marinus were investigated. The enzyme is a proton pump which has all the residues of the proton-transfer pathways found in the mitochondrial-like enzymes conserved, except for one of the key elements of the D-pathway, the helix-VI glutamate [Glu(I-286), R. sphaeroides numbering]. In contrast to what has been suggested previously as general characteristics of thermophilic enzymes, during formation of the R. marinus caa(3)-CO complex, CO binds weakly to Cu(B), and is rapidly (k(Ba) = 450 s(-1)) trapped by irreversible (K(Ba) = 4.5 x 10(3)) binding to heme a(3). Upon reaction of the fully reduced enzyme with O(2), four kinetic phases were resolved during the first 10 ms after initiation of the reaction. On the basis of a comparison to reactions observed with the bovine enzyme, these phases were attributed to the following transitions between intermediates (pH 7.8, 1 mM O(2)): R --> A (tau congruent with 8 micros), A --> P(r) (tau congruent with 35 micros), P(r) --> F (tau congruent with 240 micros), F --> O (tau congruent with 2.5 ms), where the last two phases were associated with proton uptake from the bulk solution. Oxidation of heme c was observed only during the last two reaction steps. The slower transition times as compared to those observed with the bovine enzyme most likely reflect the replacement of Glu(I-286) of the helix-VI motif -XGHPEV- by a tyrosine in the R. marinus enzyme in the motif -YSHPXV-. The presence of an additional, fifth electron in the enzyme was reflected by two additional kinetic phases with time constants of approximately 20 and approximately 720 ms during which the fifth electron reequilibrated within the enzyme.     

Pseudomonas aeruginosa cytochrome C(551): probing the role of the hydrophobic patch in electron transfer

Cytochrome c(551) from Pseudomonas aeruginosa is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of cd(1) nitrite reductase. The distribution of charged residues on the surface of c(551) is very anisotropic: one side is richer in acidic residues whereas the other shows a ring of positive side chains, mainly lysines, located at the border of an hydrophobic patch which surrounds the heme crevice. In order to map in cytochrome c(551) the surface involved in electron transfer, we have introduced specific mutations in three residues belonging to the hydrophobic patch, namely Val23-->Asp, Pro58-->Ala and Ile59-->Glu. The effect of these mutations was analyzed studying both the self-exchange rate and the electron-transfer activity towards P. aeruginosa cd(1) nitrite reductase, the physiological partner and P. aeruginosa azurin, a copper protein often used as a model redox partner in vitro. Our results show that introduction of a negative charge in the hydrophobic patch severely hampers both homonuclear and heteronuclear electron transfer.     

Water T2 relaxation in sugar solutions

1H spin-spin relaxation times of water were measured with the CPMG sequence in dilute aqueous solutions of glucitol, mannitol, glycerol, glycol, the methyl D-pyranosides of alpha-glucose, beta-glucose, alpha-galactose, beta-galactose, alpha-xylose, beta-xylose, beta-arabinose and sucrose, alpha,alpha-trehalose, beta-maltose, maltotriose and maltoheptaose. The relaxation-time dispersion was measured by varying the CPMG pulse spacing, tau. These data were interpreted by means of the Carver-Richards model in which exchange between water protons and labile solute hydroxyl protons provides a significant contribution to the relaxation. From the dependences on temperature and tau, parameters characteristic of the pool of hydroxyls belonging to a given solute were extracted by nonlinear regression, including: the fraction of exchangeable protons, P, the chemical-shift difference between water protons and hydroxyl protons, deltaomega, the intrinsic spin-spin relaxation time, T2, and the chemical exchange rate, k. These solute-specific parameters are related, respectively, to the concentration, identity, mobility and exchange life-time of the hydroxyl site. At 298 K, values of deltaomega, T2 and k were found to be of the order of 1 ppm, 100 ms and 1000 s(-1), respectively. Effects of molecular size, conformation and solute concentration were investigated. The exchange mechanism was characterised by Eyring activation enthalpies and entropies with values in the ranges 50-70 kJ mol(-1) and -10 to 60 J K(-1)mol(-1), respectively.     

Modulation of the electron redistribution in mixed valence cytochrome C oxidase by protein conformational changes

The redistribution of two electrons in the four redox centers of cytochrome c oxidase following photodissociation of CO from the CO-bound mixed valence species has been examined by resonance Raman spectroscopy. To account for both the kinetic data, obtained from 5 micros to 2 ms, and the equilibrium results, a model is proposed in which the electron redistribution is modulated by a protein conformation transition from a nascent P(1) state to a relaxed P(2) state in a time window longer than 2 ms. In this model, all six possible two-electron reduced species are considered. The high population of species with a one-electron reduced binuclear center, in which the spectrum of heme a(3) is perturbed by the redox state of Cu(B), accounts for the significant residuals in the fitting of the kinetic data with four standard spectra derived from redox species with either zero or two electrons in the binuclear center. Under equilibrium conditions, the conformational change to the P(2) state destabilizes the redox states with only one electron in the binuclear center with respect to those with either zero or two electrons. As a result, the redox equilibrium is perturbed, and the electrons are redistributed. A simulation based on the new kinetics scheme, in which the electron redistribution is modulated by the protein conformation, gives reasonable agreement with both the equilibrium and the kinetic data, demonstrating the validity of this model.