Cytochrome turnover

As noted, extensive studies of the effect of site-directed mutagenesis on proton-transfer have been carried out with Rb. sphaeroides reaction centers The most likely candidates for proton relay, the conjugate bases of the amino acids, Asp-213. Ser-223 and Glu-212. have been investigated by replacing them one at a time with amino acids which either can or cannot be protonated under physiological condition. The results of their studies on the effects of mutation as monitored by cytochrome turnover are summarized in Fig. 7. 

Work on site-directed mutagenesis involving Asp-213 was carried out by Takahashi and Wraight and by Feher and coworkers. Illumination of Asp-213->Asn-mutant reaction centers also showed an initial rapid oxidation of two cytochrome-c molecules [Fig. 7 (D), top]. This result is similar to the Ser-223->Ala-mutant-mutant, and indicates that Asp-213 is also involved in the uptake of the first proton. Subsequent cytochrome oxidation is -100 times slower and A ab(2) becomes -6000 times smaller. Replacement of Ser-223 with the acidic Glu gave a somewhat faster cytochrome turnover rate, but the effect onkAB(2)Was similar to that found by replacing it with Asn. 

Cytochrome Turnover in Native and Mutant RCs. RCs subjected to continuous illumination in the presence of cyt c and excess Q catalyze photooxidation of cyt c (monitored at 550 nm) coupled to the reduction of quinone according the cycle shown in Fig. 6. Native RCs (either Rb. sphaeroides R-26 or 2.4.1) show a rapid cyt c photooxidation (Fig. 7) with a rate constant of 200 s (at saturating cyt c and Qo concentration and light-intensity). The EQ212 mutant RCs exhibit very different kinetics (Fig. 7). They show a fast (within 3 ms) oxidation of 2.9 0.2 cyt c per RC, followed by a slower phase with a rate constant of 7 2 s". The fast oxidation of 3 cyt c indicates the rapid reduction of DQaQb to DQaQb - The slow phase in the cytochrome photooxidation indicates a block in the turnover of the quinone. The reduced turnover rate is consistent with a block in the proton uptake step(s). 

Remarkably, the fast reduction of b-563 appears to proceed the flash-induced oxidation of c-554. Reduced ferredoxin or another reductant on the acceptor side of PSI is probably responsible for the fast reduction of b-563. Relatively little cytochrome b-563 and c-554 is seen to turnover, about 25% of the content of each the mutual ratio is about 2 (measured 14 times in different preparations), indicating that for one c-554 two b-563 cytochromes turnover. The slow-rising component in the carotenoid response has a tg.s of about 35 ms (Fig. 1), about similar to tg.s of the rereduction of c-554 (30 ms), but significantly smaller than tg.s of the reoxidation of b-563 (90 ms). So, it seems unlikely that the reoxidation of b-563 is responsible for Aa518 (slow) as proposed for chloroplasts by Selak and Whitmarsh (1980). 

In be complexes bci complexes of mitochondria and bacteria and b f complexes of chloroplasts), the catalytic domain of the Rieske protein corresponding to the isolated water-soluble fragments that have been crystallized is anchored to the rest of the complex (in particular, cytochrome b) by a long (37 residues in bovine heart bci complex) transmembrane helix acting as a membrane anchor (41, 42). The great length of the transmembrane helix is due to the fact that the helix stretches across the bci complex dimer and that the catalytic domain of the Rieske protein is swapped between the monomers, that is, the transmembrane helix interacts with one monomer and the catalytic domain with the other monomer. The connection between the membrane anchor and the catalytic domain is formed by a 12-residue flexible linker that allows for movement of the catalytic domain during the turnover of the enzyme (Fig. 8a see Section VII). Three different positional states of the catalytic domain of the Rieske protein have been observed in different crystal forms (Fig. 8b) (41, 42) ... 

In addition to their proven capacity to catalyze a highly efficient and rapid reduction of O2 under ambient conditions (e.g., cytochrome c oxidase, the enzyme that catalyzes the reduction of >90% of O2 consumed by a mammal, captures >80% of the free energy of ORR at a turnover frequency of >50 O2 molecules per second per site), metalloporphyrins are attractive candidates for Pt-free cathodes. Probably the major impetus for a search for Pt-free cathodic catalysts for low temperature fuel cells is... 

Only three steps of the proposed mechanism (Fig. 18.20) could not be carried out individually under stoichiometric conditions. At pH 7 and the potential-dependent part of the catalytic wave (>150 mV vs. NHE), the —30 mV/pH dependence of the turnover frequency was observed for both Ee/Cu and Cu-free (Fe-only) forms of catalysts 2, and therefore it requires two reversible electron transfer steps prior to the turnover-determining step (TDS) and one proton transfer step either prior to the TDS or as the TDS. Under these conditions, the resting state of the catalyst was determined to be ferric-aqua/Cu which was in a rapid equilibrium with the fully reduced ferrous-aqua/Cu form (the Fe - and potentials were measured to be within < 20 mV of each other, as they are in cytochrome c oxidase, resulting in a two-electron redox equilibrium). This first redox equilibrium is biased toward the catalytically inactive fully oxidized state at potentials >0.1 V, and therefore it controls the molar fraction of the catalytically active metalloporphyrin. The fully reduced ferrous-aqua/Cu form is also in a rapid equilibrium with the catalytically active 5-coordinate ferrous porphyrin. As a result of these two equilibria, at 150 mV (vs. NHE), only <0.1%... 

Collman JP, Decreau RA, Yan Y, Yoon J, Solomon El. 2007a. Intramolecular single-turnover reaction in a cytochrome c oxidase model bearing a Tyr244 mimic. J Am Chem Soc 129 5794. 

Cholesterol transport and regulation in the central nervous system is distinct from that of peripheral tissues. Blood-borne cholesterol is excluded from the CNS by the blood-brain barrier. Neurons express a form of cytochrome P-450, 46A, that oxidizes cholesterol to 24(S)-hydroxycholesterol [11] and may oxidize it further to 24,25 and 24,27-dihydroxy products [12]. In other tissues hydroxylation of the alkyl side chain of cholesterol at C22 or C27 is known to produce products that diffuse out of cells into the plasma circulation. Although the rate of cholesterol turnover in mature brain is relatively low, 24-hydroxylation may be a principal efflux path to the liver because it is not further oxidized in the CNS [10]. 

Schrag, M.L. and Wienkers, L.C. (2005) Catalytic turnover of pyrene by CYP3 A4 evidence that cytochrome b5 directly induces positive cooperativity. Archives of Biochemistry and Biophysics, 438 (1), 21-28. 

Correia, M.A. (1991) Cytochrome P450 turnover. Methods in Enzymology, 206, 315-325. 

The influence of phenobarbital on the turnover of hepatic microsomal cytochrome b, and cytochrome P-1+50 in the rat. Biochim. Biophys. Acta (1970) 201, 20-05-... 

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. 

A single turnover study of the conversion of the heme-HO-1 complex to free biliverdin has elucidated the relative rates of the catalytic steps 129). This transient kinetic study indicates that the conversion of Fe heme to Fe verdoheme is biphasic. Electron transfer to the Fe -heme HO-1 complex occurred at a rate of 0.11 s at 4°C and 0.49 s at 25°C with a 0.1 1 ratio of NADPH-cytochrome P450 reductase to heme HO-l complex. Oxygen binding to the reduced iron was sufficiently rapid im-der the experimental conditions that the species actually monitored... 

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