The single chlorophyll a molecule in the cytochrome b6f complex: unusual optical properties protect the complex against singlet oxygen

The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.     

Characterization of long-range electron transfer in mixed-metal [zinc,iron] hybrid hemoglobins

Measurements characterizing electron transfer from a photoexcited zinc protoporphyrin triplet (3ZnP) to a ferriheme electron acceptor within the [alpha 1,beta 2] electron-transfer complex of [FeIII,Zn] hybrid hemoglobins are reported. Analytical results demonstrate that the hybrids studied are pure, homogeneous proteins with 1:1 ZnP:FeP content. Within the T quaternary structure adopted by these hybrids, the optical spectrum of a FeIIIP is perturbed by the protein environment. Room temperature kinetic studies of the rate of 3ZnP decay as a function of the heme oxidation and ligation state demonstrate that quenching of 3ZnP by FeIII(H2O)P occurs by long-range intramolecular electron transfer with rate constant kt = 100 (+/- 10) s-1 and is not complicated by spin-quenching or energy-transfer processes; results are the same for alpha(Zn) and beta(Zn) hybrids. Replacement of H2O as a ligand to the ferriheme changes the 3ZnP----FeIIIP electron-transfer rate constant, kt, which demonstrates that electron transfer, not conformational conversion, is rate limiting. However, the trend is not readily explained by simple considerations of spin-state and bonding geometry: kt decreases in the order imidazole greater than H2O greater than F- approximately CN- approximately N3-. The reverse electron-transfer process FeIIP----ZnP+ has not been observed directly but has been shown to be much more rapid, with rate constant kb greater than 10(3) s-1, consistent with the possible importance of "hole" superexchange in electron tunneling within protein complexes.     

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c.

The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of electron acceptors with the excited triplet state of Zn cytochrome c

The decay rate of the excited triplet state of Zn cytochrome c was enhanced by electron acceptors including methyl viologen and ferric complexes of cyanide, oxalate, EDTA and cytochrome c at room temperature. Ferrous compounds were several orders of magnitude less effective than the respective ferric form in quenching the phosphorescence. In the presence of ferricytochrome c and ferricyanide the semilogarithmic plots of the decay curve showed an anomalous decay profile in which the rate of interaction appeared to accelerate after excitation. One explanation is that the quenching process was accelerated by a conformational change of the polypeptide chain around the excited triplet state porphyrin. Another explanation is that quenching occurs via an intermediate.     

Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase.

The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxidase were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines. Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the heme c and the hemes a and a3. The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2. Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket. In cytochrome c oxidase, the structural changes in both hemes refer to more subtle perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states. These changes are qualitatively different to those observed for cytochrome c oxidase upon poly-l-lysine binding. The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively. The spectroscopic studies were paralleled by steady-state kinetic measurements of the same protein combinations. The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants. The complex stability decreases in the order: wild-type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine). This order is not exhibited by the conformational equilibria. The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cytochrome c that is defined by the lysyl residues at positions 72, 73 and 79. On the other hand, the conformational changes in cytochrome c oxidase were found to be independent of the identity of the bound cytochrome c variant. The maximum rate constants determined from steady-state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis leads to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1. These results can be interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.     

Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm. On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c. Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1). On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism.     

Triplet-state kinetics of Zn-porphyrin cytochrome c in micellar media. Measurement of intermicellar exchange rates

The interactions of protein molecules with surfactant assemblies in aqueous and hydrocarbon media have been studied via the triplet-state kinetics of Zn-porphyrin cytochrome c in solutions containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] or a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. In aqueous solution, the observed triplet state decay is single exponential with a lifetime of 8 ms. In aqueous solutions of AOT and in AOT-reversed micellar solutions, biexponential triplet state decays were observed, indicating that interactions between the surfactant and the protein occur, resulting in a change in protein conformation near the porphyrin ring. In CTAB-reversed micellar solutions, quenching of the Zn-porphyrin cytochrome c triplet state by ferricyanide and methyl viologen was studied. Because the quenching is exchange-limited under the conditions used, the exchange rate constants for the water pools can be obtained from these experiments. The observed exchange rate constants are in the range (1-5) x 10(7) M-1 S-1, depending on the water content of the reversed micelles and on the type of quencher used. These values are three orders of magnitude lower than the calculated collision rate of the reversed micelles.     

Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.     

Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: a fluorescence quenching study on the ionic strength dependence of the interaction

The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.     

Cytochrome c at charged interfaces. 2. Complexes with negatively charged macromolecular systems studied by resonance Raman spectroscopy

We have analyzed the structure of cytochrome c (cyt c) bound in a variety of complexes in which negatively charged molecular groups interact with the positively charged binding domain around the heme crevice of cyt c. Using resonance Raman spectroscopy, we could demonstrate that these interactions induce the same conformational changes as they were observed in the surface-enhanced resonance Raman experiments of cyt c adsorbed on the Ag electrode [Hildebrandt & Stockburger (1989) Biochemistry (preceding paper in this issue)]. When cyt c is bound to (As4W40O140)27-, state II is stabilized, whereas in complexes with phosvitin and cytochrome b5 state I is formed. The complexes with phospholipid vesicles and inverted micelles reveal a mixture of both states. It is suggested that these systems as well as cyt c adsorbed on the Ag electrode may be regarded as model systems for the physiological complexes of cyt c with cytochrome oxidase and cytochrome reductase. On the basis of our findings it is proposed that the biological electron-transfer reactions are controlled by electric field induced conformational transitions of cyt c upon complex formation with its physiological redox partners.     

Diffusion-limited rates for monoclonal antibody binding to cytochrome c.

The kinetic and spectroscopic changes accompanying the binding of two monoclonal antibodies to the oxidized form of horse heart cytochrome c have been investigated. The two epitopes recognized by the antibodies are distinct and noninteracting: antibody 2B5 binds to native cytochrome c near a type II turn (residue 44) while antibody 5F8 binds on the opposite face of the protein near the amino terminus of an alpha-helical segment (residue 60). Antibody-cytochrome c binding obeys a simple bimolecular reaction mechanism with second-order rate constants approaching those expected for diffusion-limited protein-protein interactions. The association rate constants have small activation enthalpies and are inversely dependent on solvent viscosity, as expected for diffusion-controlled reactions. There is a moderate ionic strength dependence of the rate of association between the 2B5 antibody and cytochrome c, with the rate constant increasing about 4-fold as the ionic strength is varied between 0.14 and 0 M. Comparison of the rates for antibody-cytochrome c complex formation for binding to the reduced-native, oxidized-native, and alkaline conformations shows that for MAb 2B5 the forward rate constant depends slightly on cytochrome c conformation. Investigation of the pH-induced transition between the native and alkaline conformational states for free cytochrome c and for antibody-cytochrome c complexes shows that antibody binding stabilizes the native form of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein dynamics: imidazole and 2-mercaptoethanol binding to the Rhodobacter capsulatus cytochrome c(2) mutant,glycine 95 proline

The Class I c-type cytochromes can bind exogenous ligands in the oxidized state, with the kinetics of ligand binding providing information on naturally occurring intramolecular dynamics. Typically, nitrogenous bases are used as ligands; however, it is less well known that 2-mercaptoethanol (BME), a commonly used cytochrome reducing agent, can form a complex with the heme. To better understand the cytochrome-mercaptan interaction, we have investigated the kinetics of binding of BME to wild type and mutants of Rhodobacter capsulatus cytochrome c(2) and to horse cytochrome c. Complex formation with the G95P mutant is apparent from the formation of a green color and a shift in the Soret peak to 418 nm from 410 nm upon addition of BME. Unlike horse cytochrome c and wild-type R. capsulatus cytochrome c(2), G95P permits the kinetics of formation of the BME-G95P complex to be measured since complex formation and reduction kinetics can be resolved. The affinity constant for the binding of BME to mutant G95P was strong ( approximately 1.5 x 10(5)M(-1)) and the kinetics of formation of the BME-G95P complex were found to undergo a change in rate-limiting step consistent with a concentration-independent protein rearrangement (68s(-1)) followed by second-order binding of BME ( approximately approximately 1.3 x 10(5)M(-1)s(-1)). The most remarkable characteristic of mutant G95P is the relatively large amount of high-spin species in equilibrium with the low- spin form, which can be estimated to be approximately 3% at pH 7. The BME binding kinetics, coupled with the kinetics of imidazole binding to G95P, allow us, for the first time, to specify all four rate constants describing the ligand binding reaction. Moreover, we can use the kinetic results to estimate the rate constants for ligand binding with the wild-type cytochrome c(2). This has also allowed us to quantify and more fully interpret cytochrome dynamics.     

Size exclusion chromatographic analysis of polyphenol-serum albumin complexes

Formation of water-soluble polyphenol-protein complexes was investigated by size-exclusion chromatography (SEC). The combination of (-)-epigallocatechin gallate (EGCG) and bovine serum albumin (BSA), which did not form a precipitate after the solutions were mixed, showed an SEC peak due to complex formation 2-24 h after mixing. Peak size of the complex varied with time, suggesting slow change of the conformation of the protein accompanied by complexation. Formation of the complex was substantiated by ultrafiltration of the mixture; the complex did not pass through a membrane with a 100,000 nominal molecular weight limit (NMWL). The SEC profile varied with the combination of compounds. The peaks due to the complexes showed that the apparent value of the number average molecular weight (M(n)) of the EGCG-BSA complex was 2.8x10(5), while that of a pentagalloylglucose (PGG)-BSA complex was 9.5x10(5) under the conditions used. Dimeric hydrolyzable tannins, oenothein B and cornusiin A, also caused changes in the SEC profile of BSA, although the combinations did not show peaks attributable to formation of such large complexes observed for EGCG and PGG. Procyanidin B3 and (+)-catechin did not cause changes in the SEC profile of BSA. With cytochrome c, EGCG did not show any chromatographic changes.     

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.     

Transient kinetics of redox reactions of flavodoxin: effects of chemical modification of the flavin mononucleotide prosthetic group on the dynamics of intermediate complex formation and electron transfer

The effects of structural modifications of the flavin mononucleotide (FMN) prosthetic group of Clostridium pasteurianum flavodoxin on the kinetics of electron transfer to the oxidized form (from 5-deazariboflavin semiquinone produced by laser flash photolysis) and from the semiquinone form (to horse heart cytochrome c by using stopped-flow spectrophotometry) have been investigated. The analogues used were 7,8-dichloro-FMN, 8-chloro-FMN, 7-chloro-FMN, and 5,6,7,8-tetrahydro-FMN. The ionic strength dependence of cytochrome c reduction was not affected by chlorine substitution, although the specific rate constants for complex formation and decay were appreciably smaller. On the other hand, all of the chlorine analogues had the same rate constant for deazariboflavin semiquinone oxidation. The rate constants for tetrahydro-FMN flavodoxin semiquinone reduction of cytochrome c were considerably smaller than those for the native protein. The implications of these results for the electron-transfer mechanism of flavodoxin are discussed.     

Effects of amino acid replacements in yeast iso-1 cytochrome c on heme accessibility and intracomplex electron transfer in complexes with cytochrome c peroxidase

The kinetics of reduction of wild type and several site-specific mutants of yeast iso-1 cytochrome c (Arg-13----Ile, Gln-16----Ser, Gln-16----Lys, Lys-27----Gln, Lys-72----Asp), both free and in 1:1 complexes with yeast cytochrome c peroxidase, by free flavin semiquinones have been studied. Intramolecular one-electron transfer from the ferrous cytochromes c to the H2O2-oxidized peroxidase at both low (8 mM) and high (275 mM) ionic strengths was also studied. The accessibility of the cytochrome c heme within the electrostatically stabilized complex and the rate constants for intramolecular electron transfer at both low and high ionic strength are highly dependent on the specific amino acids present at the protein-protein interface. Importantly, replacement by uncharged amino acids of Arg or Lys residues thought to be important in orientation and/or stabilization of the electron-transfer complex resulted in increased rates of electron transfer. In all cases, an increase in ionic strengths from 8 to 275 mM also produced increased intramolecular electron-transfer rate constants. The results suggest that the electrostatically stabilized 1:1 complex is not optimized for electron transfer and that by neutralization of key positively charged residues, or by an increase in the ionic strength thereby masking the ionic interactions, the two proteins can orient themselves to allow the formation of a more efficient electron-transfer complex.     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

Voltammetry of a "protein on a rope"

A periplasmic electron-transfer protein, cytochrome c(555)(m) from Aquifex aeolicus contains a 62-residue N-terminal extension by which it is anchored to the membrane--most probably via a thioester bond to its N-terminal cysteine. This linker can act as a "rope" to tether the protein close to its reaction partners. Mimicking this principle, a recombinant cytochrome c(555)(m), expressed in Escherichia coli, has been attached covalently to a gold electrode modified with 6-mercaptohexan-1-ol. The "tethered" cytochrome c(555)(m) displays remarkably fast electron-transfer kinetics, with an electrochemical exchange rate constant k(0) of 1.4 x 10(4) s(-1). The results show that fast electron transfer is associated with weak interactions: importantly, the tethered cytochrome can explore many different orientations without escaping into solution.     

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.