Electron transfer kinetics involving cytochrome

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... 

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. 

Many kinetic studies have been carried out on the reactions of cytochrome c. This work, as is the case for other electron-transfer proteins, has followed two general courses. One approach involves the study of reactions of cytochrome c with inorganic and organic reagents and with isolated electron-transfer proteins. The second approach has involved the use of intact or partially disrupted mitochondrial or photosynthetic electron-transfer systems. 

The reaction between HiPIP and cytochrome c or bacterial cytochromes784 involves specific sites on both proteins, although no kinetic evidence for association was found. Reaction between Chr. vinosum and Rhodopseudomonas gelatinosa HiPIPs with modified cytochrome c (trinitro-phenyllysine-13)785 was more rapid than for the unmodified cytochrome c since modification of Lys-13 destabilizes the heme crevice, and because the hydrophobic TNP group facilitates electron transfer by interacting with a hydrophobic region of the HiPIP. 

Before the chemical identity of the secondary electron acceptor and the reaction mechanism involved were known. Parson obtained some useful information indirectly from spectro-kinetic studies using a double-flash arrangement. Parson used a pair of laser flashes spaced a few microseconds apart to excite the chromatophores of Chromatium vinosum and found that while the first flash elicited photooxidation of P870, the second flash did not cause another photooxidation even though the photooxidized P870 " has been re-reduced by the endogenous, c-type cytochrome within -2 /js and presumably ready to undergo another photooxidation, provided there had been electron transfer from Qa Io Qb, i.e.,... 

It is of note that Kaminskaya, Konstantinov and Shuvalov also examined in detail the kinetics of low-temperature photooxidation of all four cytochromes in Rp. viridis reaction centers and established the complete heme sequence as HI, LI, H2, L2, as shown in Fig. 7, thus putting into doubt the earlier model involving two parallel H/L electron-transfer pathways, as suggested by the model in Fig. 2. 

X 10 M s and was 3.1 x 10 M s" at 25°C, pH 7.0 and ionic strength of 1.0 . Kinetic data was interpreted in terms of a mechanism of electron transfer from chromium(II) involving attack of Cr(II) adjacent to the Fe(III) center Analysis of the one-to-one chromium(III) cytochrome c complex revealed that the chromium(III) cross-linked two peptide fragments located in the heme.crevice by binding to tyrosine 67 and asparagine 52 The chromium(III) bound to reduced cytochrome c did not affect the ability of the protein to be reoxidized with ferricyanide and then to be reduced with dithionite . The chromium complex was oxidized by cytochrome oxidase at the same rate as the untreated ferrocytochrome c, however, the rate of reduction of the chromium complex by bovine heart submitochondrial particles was slower than that of untreated ferricytochrome c Thus, the binding of chromium(III) to cytochrome c appears to selectively inhibit its function in certain electron transfer reactions. 

Electrons are transferred by the respiratory electron chain to a cytochrome b which then donates electrons to the Mo-protein (nitrate reductase). By using reduced viologens it should be possible to determine the kinetics of nitrate reduction by the nitrate reductase however, no information on this aspect is currently available. The sensitivity of nitrate reduction to inhibition by cyanide is considered to be due to association of the inhibitor with molybdenum residues in the nitrate reductase (Enoch and Lester, 1975). Cytochrome b involved in nitrate reduction does not bind cyanide. 

---