Cytochrome electron transfer kinetics

Steady state kinetics and protein-protein binding measurements have also been reported for the interaction of these mutant cytochromes with bovine heart cytochrome c oxidase [120]. The binding of cytochrome c variants to the oxidase occurred with increasing values of Kj in the order He (3 x 10 Mol L ) < Leu = Gly < wild-type < Tyr < Ser (3 x 10 molL ). Steady-state kinetic analysis indicated that the rate of electron transfer with cytochrome c oxidase increased in the order Ser < He < Gly < Leu < Tyr < wild-type, an order notably different from that observed for a related analysis of the oxidation of these mutants by cytochrome c peroxidase [85]. This difference in order of mutant turnover by the oxidase and peroxidase may arise from differences in the mode of interaction of the cytochrome with these two enzymes. 

As part of a subsequent study concerning primarily second-site revertant yeast iso-l-cytochrome c variants, Hazzard et al. evaluated the effect of converting Lys-72 to an aspartyl residue by site-directed mutagenesis on the electron transfer kinetics of the cytochrome c-cytochrome c peroxidase complex [136]. Lys-72 was of interest for this purpose, because it is involved in the hypothetical model for the complex formed by these two proteins that was proposed by Poulos and Kraut on the basis of molecular graphics docking [106]. In these... 

Recent structural studies and electron transfer kinetic experiments focus on structures in which a site-specific covalent crosslink between cytochrome c and cytochrome c peroxidase subunits exists. One of these used site-directed mutagenesis to form a disulfide bond between a V197C mutant CcP and an A81C... 

Reaction of Cytochrome cimu with Tris(oxalato)cobalt(III) The cytochrome c protein was also used as reductant in a study of the redox reaction with tris (oxalato)cobalt(III).284 Selection of the anionic cobalt(III) species, [Conl(ox)3]3 was prompted, in part, because it was surmised that it would form a sufficiently stable precursor complex with the positively charged cyt c so that the equilibrium constant for precursor complex formation (K) would be of a magnitude that would permit it to be separated in the kinetic analysis of an intermolecular electron transfer process from the actual electron transfer kinetic step (kET).2S5 The reaction scheme for oxidation of cyt c11 may be outlined ... 

Durham BD, Pan LP, Hahm S, Long J, Millett F. Electron-transfer kinetics of singly labelled ruthenium(II) polypyridine cytochrome c derivatives. In Johnson MK, King RB, Kurtz DM, Kutal C, Norton ML, Scott RA (eds), Electron Transfer in Biology and the Solid States Inorganic compounds with unusual properties. ACS Advances in Chemistry Series. Washington DC American Chemical Society, 1990 180-93. 

Wang K, Zhen Y, Sadoski R, et al. Definition of the interaction domain for cytochrome c on cytochrome c oxidase. II. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants. / Biol Chem 1999 274 38042-50. 

Lee JC, Chang I-J, Gray HB, Winkler JR. The cytochrome c folding landscape revealed by electron-transfer kinetics. J Mol Biol 2002 320 159-64. 

The electron transfer from cytochrome c to O2 catalyzed by cytochrome c oxidase was studied with initial steady state kinetics, following the absorbance decrease at 550 nm due to the oxidation of ferrocyto-chrome c in the presence of catalytic amounts of cytochrome c oxidase (Minnart, 1961 Errede ci a/., 1976 Ferguson-Miller ei a/., 1976). Oxidation of cytochrome c oxidase is a first-order reaction with respect to ferrocytochrome c concentration. Thus initial velocity can be determined quite accurately from the first-order rate constant multiplied by the initial concentration of ferrocytochrome c. The initial velocity depends on the substrate (ferrocytochrome c) concentration following the Michaelis-Menten equation (Minnart, 1961). Furthermore, a second catalytic site was found by careful examination of the enzyme reaction at low substrate concentration (Ferguson-Miller et al., 1976). The Km value was about two orders of magnitude smaller than that of the enzyme reaction previously found. The multiphasic enzyme kinetic behavior could be interpreted by a single catalytic site model (Speck et al., 1984). However, this model also requires two cytochrome c sites, catalytic and noncatalytic. 

Ruthenium complexes are excellent reagents for protein modification and electron-transfer studies. Ru +-aquo complexes readily react with surface His residues on proteins to form stable derivatives [20, 21]. Low-spin pseudo-octahedral Ru-complexes exhibit small structural changes upon redox cycling between the Ru + and Ru + formal oxidation states [3, 22]. Hence, the inner-sphere barriers to electron transfer (Ai) are small. With the appropriate choice of ligand, the Ru + + reduction potential can be varied from <0.0 to >1.5 V versus NHE [23]. Ru-bpy complexes bound to Lys and Cys residues have been employed to great advantage in studies of protein-protein ET reactions. The kinetics of electron transfer in cytochrome 65/cytochrome c [24], cytochrome c/cytochrome c peroxidase [12], and cytochrome c/cytochrome c oxidase [25] complexes have been measured with the aid of laser-initiated ET from a Ru-bpy label. 

Figure 2. Electron transfer kinetics of cytochrome c oxidation in Chromatium vinosum [4] and Rhodopseudomonas viridis [16] display temperature independence at low temperature, a herald of tunneling. The early Chromatium data were analyzed as a single phase, while the Rp. viridis data were analyzed into three phases, dominated by very fast (VF) and fast (F) phases at high temperatures, and dominated by slow (S) phase at low temperatures.

Cytochrome oxidase catalyses electron transfer from cytochrome c to dioxygen, reducing the latter to water without release of intermediates. The maximal electron transfer activity may reach 400 (moles of cytochrome c oxidised per second per mole of cytochrome aa ) in optimal conditions at pH 7 and 25°C, both in situ and in detergent solution, although much lower activities are often encountered in the latter case (see Ref. 99). In mitochondria, respiration with natural substrates proceeds at much lower rates (cf.. Section 2.3). The kinetic capacity of cytochrome oxidase greatly exceeds demands for reasons not understood at present. 

The present view is that cytochrome a is the acceptor of electrons from cytochrome c, but that a simple linear electron-transfer sequence from cytochrome a to Cua and then to the cytochrome 03/Cub centre is unlikely. Instead the sequence shown in equation (63) holds, where cytochrome a is in rapid equilibrium with Cua. These views depend largely upon pre-steady-state kinetics of the redox half reactions of the enzyme with its two substrates, ferrocytochrome c and O2. However, these conclusions are not in accord with kinetic studies under conditions when both substrates are bound to the enzyme, and which show maximal rates of electron transfer from cytochrome c to O2. In particular some of the cytochrome c is oxidized at a faster rate than a metal centre in the oxidase. In contrast, at high ionic strength conditions, where the cytochrome c and the cytochrome oxidase are mainly dissociated, oxidation of cytochrome c occurs only slowly following the complete oxidation of the oxidase. These results for the fast oxidation of cytochrome c have been interpreted in terms of direct electron transfer from cytochrome c to the bridged peroxo intermediate involving 03 and Cub, or to a two-electron transfer to O2 from cytochromes a and 03 during the initial phase of the reaction. 

R.LC. (2009) Electron transfer kinetics of cytochrome c probed by bme-resolved surface-enhanced resonance Raman spectroscopy. Journal of Physical Chemistry B, 113, 2492-2497. 

Over the past several years, we have developed a technique that has proven extremely valuable in the study of electron transfer between redox sites in metalloproteins. We have reported kinetic studies of the reaction of cytochrome c with cytochrome c peroxidase (i-3), cytochrome oxidase (4), cytochrome bs (5, 6) plastocyanin (7), and cytochrome Ci (8). In addition, we have been able to show (9,10) that intramolecular electron transfer in cytochrome bs covalently... 

El Kasmi, A., Wallace, J. M., Bowden, E. R, Binet, S. M., Linderman, R. J. (1998). Con-trolling interfacial electron-transfer kinetics of cytochrome c with mixed self-assembled monolayers, J. Am. Chem. Soc., 120 225. 

Heterogeneous Electron Transfer Kinetic Parameters for Cytochrome c System... 

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