Role of cytochrome b5 in catalysis by cytochrome P450 2B4

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.     

Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b6-f complex

The chloroplast cytochrome b6-f complex, incorporated into phospholipid vesicles, shows proton translocation with an observed H+/e- ratio of approximately 2. The oxidation-reduction behavior of cytochrome b6 during electron transport from duroquinol to plastocyanin is affected by incorporation. The most obvious effect of incorporation is an increase in the duration of a steady-state level of cytochrome b6 that persists during electron transport. Reagents that decrease activity increase the duration of the steady state while reagents that stimulate activity decrease this time. Uncoupling conditions yield cytochrome kinetics similar to those in the unincorporated complex. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone and 5-n-undecyl-4,7-dioxobenzothiazole inhibited reduction of cytochrome b6 in the incorporated complex, but this apparent inhibition was due to a rapid oxidation of the cytochrome by these compounds.     

The "cytochrome b5 fold": structure of a novel protein superfamily

Selective proteolysis allows the isolation of a heme-binding fragment spectrally similar to microsomal cytochrome b₅ from both baker's yeast flavocytochrome b₂ (a flavohemoprotein) and liver sulfite oxidase (a molybdoprotein). The amino acid sequences of these two fragments have been published separately (Guiard &; Lederer, 1976,1979). We present in this paper an alignment of those sequences with that of microsomal cytochrome b₅. The structural consequences of the similarity between the three primary structures are discussed in the light of the cytochrome b₅ three-dimensional model (Mathews et al., 1971,1972,1975; Mathews &; Czerwinski, 1976).It is concluded that the three heme-binding proteins are in all probability the products of a divergent evolution from a common ancestor and that they must present a basically similar backbone with some surface alterations. We propose to name this backbone the “cytochrome b₅ fold”. The comparison of the three proteins suggests hypotheses concerning the molecular surface areas involved in the recognition of cytochrome c (the common acceptor) and of the respective reductase (flavo- or molybdoprotein).In addition, our results suggest that at some point in evolution, several copies of an initial hemoprotein gene were formed in the cellular genome. Subsequently, one copy was fused with the gene for another function: a flavoreductase in yeast cells or a molybdoreductase in hepatic cells.     

Age-dependent decay of cytochrome b5 and cytochrome b5 reductase in human erythrocytes.

Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.