Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin.

1. A detailed study of cytochrome C oxidse activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome C concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholipid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD2 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2...     

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.     

Do evolutionary changes in cytochrome c structure reflect functional adaptations?

Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.     

Low-temperature studies of electron transfer between different cytochromes c and cytochrome c oxidase.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.     

ATP-induced spectral changes in cytochrome c oxidase. A kinetic investigation.

Mixing ATP with soluble oxidized cytochrome c oxidase induces a spectral perturbation in the Soret region of the enzyme. This spectral perturbation is observed at ATP concentrations similar to those found to modulate the catalytic activity of cytochrome c oxidase [Malatesta, Antonini, Sarti & Brunori (1987) Biochem. J. 248, 161-165]. The process is reversible and corresponds to a simple binding with Kd = 0.2 mM at 25 degrees C. The absorbance change follows a first-order time course, and analysis of the ATP-concentration-dependence indicates the presence of a rate-limiting monomolecular step that governs the process. From the temperature-dependence of this process, studied at saturating concentrations of ATP, an activation energy of 44 kJ/mol (10.6 kcal/mol) was measured. The spectral perturbation also occurs when cytochrome c oxidase is reconstituted into artificial phospholipid vesicles, with equilibria and kinetics similar to those observed with the soluble enzyme. Mixing ATP with soluble oxidized cyanide-bound cytochrome c oxidase induces a different spectral perturbation, and the apparent affinity of ATP for the enzyme is substantially increased. There is no absolute specificity for ATP, because EGTA, inositol hexakisphosphate, sulphate and phosphate are all able to induce an identical spectral perturbation with the same kinetics, although the value of the apparent Kd is different for the various anions. The presence of Mg2+ ions decreases, in a saturation-dependent fashion, the apparent affinity of cytochrome c oxidase for ATP. The absorbance change can be correlated to the displacement of the Ca2+ bound to cytochrome c oxidase.     

Interaction of cytochrome c with cytochrome c oxidase: an understanding of the high- to low-affinity transition

The steady-state kinetics of high- and low-affinity electron transfer reactions between various cytochromes c and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparations were studied spectrophotometrically and polarographically. The dissociation constants for the binding of the first and second molecules of horse cytochrome c (I = 15 mM) are 5.10(-8) M and 1.10(-5) M, respectively, close to the spectrophotometric Km values and consistent with the controlled binding model for the interaction between cytochrome c and cytochrome oxidase (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 346-351) which postulates that the binding of a second molecule of cytochrome c weakens that of the first, resulting in low-affinity kinetics. While the Km of the polarographically assayed high-affinity reaction is comparable to that observed spectrophotometrically, the low-affinity Km is over an order of magnitude smaller and cannot be attributed to the binding of a second molecule of cytochrome c. Increasing the viscosity has no effect on the Vmax of the low-affinity reaction assayed polarographically, but increases the Km. Thus, the transition from high- to low-affinity kinetics is dependent on the frequency of productive collisions, as expected for a hysteresis model ascribing the transition to the trapping of the oxidase in a primed state for turnover. At ionic strengths above 150 mM, the rate of cytochrome c oxidation decreases without any correlation to the calculated net charge of the cytochrome c, indicating rate-limiting rearrangement of the two proteins in proximity to each other.     

Binding and oxidation of mutant cytochromes c by cytochrome-c oxidase

Mutation of conserved Phe-82 of yeast iso-1 cytochrome c to Tyr, Gly, Ser, Leu, or Ile affects binding to and reaction with cytochrome-c oxidase from beef heart. The observed changes of binding and kinetic constants reflect mutation-induced rearrangements in the heme vicinity brought about by the replacement of Phe-82. Such conformational rearrangements are also revealed by altered circular dichroism spectra of the oxidase-bound mutant cytochromes c. Variations in Km for cytochrome c oxidation do not parallel variations in Kd, the dissociation constant for binding of cytochrome c to the oxidase. This observation does not support an enzymatic mechanism in which the rate of cytochrome c oxidation is governed by product dissociation.     

Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

1. Despite the same methionine-sulfur:heme-iron:imidazole-nitrogen hemochrome structure observed by x-ray crystallography in four of the seven c-type eukaryotic and prokaryotic cytochromes examined, and the occurrence of the characteristic 695 nm absorption band correlated with the presence of a methionine-sulfur:heme-iron axial ligand in all seven proteins, they fall into two distinct classes on the basis of their EPR and optical spectra. The horse, tuna, and bakers' yeast iso-1 cytochromes c have a predominant neutral pH EPR form with g1=3.06, g2=2.26, and g3=1.25, while the bakers' yeast iso-2 and Euglena cytochromes c, the Rhodospirillum rubrum cytochrome c2, and the Paracoccus denitrificans cytochrome c550 all have a predominant neutral pH EPR form with g1=3.2, g2=2.05, and g3=1.39. The ferricytochromes with g1=3.06 have a B-Q splitting that is approximately 150 cm-1 larger than the ferricytochromes with g1=3.2. 2. Each of the cytochromes displays up to four low spin EPR forms that are in pH-dependent equilibrium and can all be observed at near neutral pH. As the pH is raised the predominant neutral pH form is converted into two forms with g1=3.4 and g1=3.6, identified by comparsion with model compounds and other heme proteins as epsilon-amino:heme-iron:imidazole and bis-epsilon-amino:heme-iron ferrihemochromes, respectively. 3. The pK for the conversion of the predominant neutral pH EPR form into the alkaline pH forms is the same as the pK for the disappearance of the 695 nm absorption band for the cytochromes, even though these pK values range over 2 pH units. This confirms that the g1=3.06 and g1=3.2 forms contain the methionine-sulfur:heme-iron axial ligand while the g1=3.4 and the g1=3.6 forms do not. 4. At extremes of pH, the horse and bakers' yeast iso-1 proteins display several high and low spin forms that are identified, showing that a variety of protein-derived ligands will coordinate to the heme iron including methionine and cysteine sulfur, histidine imidazole, and lysine epsilon-amine. 5. The spectrum of horse cytochrome c with added azide, cyanide, hydroxide, or imidazole as axial ligands has also been examined. 6. From a comparison of the EPR and optical spectral characteristics of these groups of cytochromes with model compounds, it is suggested that the difference between them is due to a change in the hydrogen bonding or perhaps even in the protonation of N-1 of the heme iron-bound histidine imidazole.     

ADP increases the affinity for cytochrome c by interaction with the matrix side of bovine heart cytochrome c oxidase

The effect of intraliposomal ADP and ATP on the kinetics of cytochrome c oxidation in reconstituted bovine heart cytochrome c oxidase was measured by the photometric and polarographic method: 1. Intraliposomal ADP decreases and intraliposomal ATP increases the Km for cytochrome c when measured by the photometric assay under uncoupled conditions. 2. The above described effects are not obtained when the kinetics are measured with the polarographic assay. 3. Extraliposomal ATP increases the Km for cytochrome c similar to intraliposomal ATP, but this effect is measured with both methods of assay. 4. Under coupled conditions only a small decrease of the Km for cytochrome c by intraliposomal ADP is found.     

Intraliposomal nucleotides change the kinetics of reconstituted cytochrome c oxidase from bovine heart but not from Paracoccus denitrificans

Isolated cytochrome c oxidases of P. denitrificans and bovine heart were reconstituted in liposomes and the kinetics of cytochrome c oxidation were measured in the presence and absence of nucleotides either inside or outside of proteoliposomes, and after photolabelling with 8-azido-ATP. Intraliposomal ATP increases and ADP decreases the kinetics of ferrocytochrome c oxidation of the bovine but not of the Paracoccus enzyme. Extra-liposomal ATP and ADP increase the Km for cytochrome c of both enzymes, but ATP acts at lower concentrations than ADP. The increase of the Km for cytochrome c is obtained in coupled as well as in uncoupled proteoliposomes. Photolabelling with 8-azido-ATP of the reconstituted Paracoccus enzyme also increases the Km for cytochrome c which is completely prevented if ATP but not if ADP is present during illumination as was found with reconstituted cytochrome c oxidase from bovine heart. The data suggest a specific interaction of ATP and ADP with nuclear-coded subunits of bovine heart cytochrome c oxidase from the matrix side, because the effects are not found with the Paracoccus enzyme, which lacks these subunits.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.     

Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.     

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.     

The effect of oxygen concentration on the steady-state kinetics of the solubilized cytochrome c oxidase.

1. The steady-state kinetics of ascorbate oxidation as a function of oxygen concentration was measured with a solubilized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparation. 2. Linear double reciprocal plots were obtained at various fixed concentrations of ascrobate, cytochrome c and cytochrome aa3. 3. The results are interpreted in terms of an oxidase model similar to that put forward by Minnaert in 1961 (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23-34). 4. The Km for oxygen at infinite cytochrome c concentration is 0.95 muM and the intramolecular rate constant for the transfer of electrons from cytochrome c to cytochome aa3 is 400 s(-1). According to the model, this implies that the second order rate constant for the reaction between oxygen and the oxidase is 9.5 X 10(7)M(-1)-s(-1).     

Half reduction potentials and oxygen affinity of the cytochromes of Pseudomonas carboxydovorans

Abstract The midpoint redox potentials (E'₀) of the cytochromes of Pseudomonas carboxydovorans have been studied by means of coupled spectrum deconvolution and potentiometric analysis. Membranes of cells grown on different substrates (CO; H₂+ CO₂; or pyruvate) contained cytochromes with similar absorption peaks and redox potentials. The cytochromes of the CO-sensitive main electron pathway of the respiratory chain revealed redox potentials in the same range as mitochondrial cytochromes (cytochrome b -555, about −20 mV; cytochrome c and cytochrome a , about +220 mV). For the cytochromes of the CO-insensitive alternative electron pathway, which allows uninhibited growth and respiration in the presence of high concentrations of CO, redox potentials of approx. +50 mV (cytochrome b -558) and −11 to −215 mV (cytochrome b -561) were determined. Cytochrome [ib-561], earlier proposed as the alternative terminal oxidase o in this organism, was shown to possess the lowest half reduction potential of all the cytochromes present in the cells. Measurements of the apparent K _m value for oxygen revealed a low affinity of cytochrome a ( K _m/ 5 υ M O₂) and a very high affinity of the CO-insensitive oxidase ( K _m < 0.5 μ M O₂). The high affinity to oxygen might be responsible for the CO-insensitivity of this unusual cytochrome o .     

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.     

Replacement of the invariant lysine 77 by arginine in yeast iso-1-cytochrome c results in enhanced and normal activities in vitro and in vivo

Oligonucleotide-directed mutagenesis of the yeast Saccharomyces cerevisiae was used to generate an abnormal iso-1-cytochrome c having an Arg-77 replacement of the normal Lys-77; this Lys-77 residue is evolutionarily conserved in most eukaryotic cytochromes c and is trimethylated in fungal and plant cytochromes c. Examination of strains having a single chromosomal copy of the gene encoding the Arg-77 protein indicated that the altered protein was synthesized at the normal rate and that it had normal or near normal activity in vivo. Examination of enzymatic activities in vitro with cytochrome b2, cytochrome c peroxidase, and cytochrome c oxidase indicated that the altered iso-1-cytochrome c has equal or enhanced catalytic efficiencies. Thus, replacement of the evolutionarily conserved residue Lys-77 produces no or only minor effects both in vivo and in vitro.     

Effect of specific lysine modification on the reduction of cytochrome c by succinate-cytochrome c reductase.

The reduction of cytochrome c by succinate-cytochrome c reductase was studied at very low cytochrome c concentrations where the reaction between cytochrome c1 and cytochrome c was rate limiting. The rate constant for the reaction was found to be independent of ionic strength up to 0.1 M chloride, and to decrease rapidly at higher ionic strength, suggesting that the interaction between cytochrome c1 and cytochrome c was primarily electrostatic. The reaction rates of cytochrome c derivatives modified at single lysine residues to form trifluoroacetylated or trifluoromethylphenylcarbamylated cytochromes c were studied to determine the role of individual lysines in the reaction. None of the modifications affected the reaction at low ionic strength, but at higher ionic strength the reaction rate was substantially decreased by modification of those lysines surrounding the heme crevice, lysine-8, -13, -27, -72, and -79. Modification of lysine-22, -25, -55, -99, and -100 had no effect on the rate. These results indicate that the binding site on cytochrome c for cytochrome c1 overlaps considerably with that for cytochrome oxidase, suggesting that cytochrome c might undergo some type of rotational diffusion during the electron-transport process.     

Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase

Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways.     

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.