Untitled

Abstract

We have shown earlier that microsomal cytochrome b5 can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b5, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b5. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b5, which are located at the site responsible for interaction with electron transfer partners, we used site-directed mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b5 were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b5 mutants were purified. Addition of the truncated forms of cytochrome b5 (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b5 in its interaction with cytochrome P450scc. However, full-length cytochrome b5 and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b5 stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b5 are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b5 about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b5 is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b5 appear to participate in electrostatic interaction with microsomal type cytochrome P450.

Interaction of Apo-cytochrome b5 with Cytochromes P4503A4 and P45017A: Relevance of Heme Transfer Reactions

“…Recently, the heterologous expression of HMW-holo-b 5 in microorganisms has been achieved, allowing the production of large amounts of the different forms of recombinant b 5 (33,34,(76)(77)(78)(79)(80)(81)(82)(83). This approach has been used to study, for example, the role of specific amino acids of b 5 by sitedirected mutagenesis (84,85), the mechanism of interaction of the C-terminal sequence of b 5 with membrane structures (86), and also the mechanism of electron transfer (87).…”

“…Immunoblotting Analysis. Immunoblotting analysis was carried out as described previously (34), using antibodies against rat liver microsomal HMW-b 5 . Immunoblotting analysis of the His67Ala mutant of b 5 was used to estimate the level of expression of the mutant protein in E. coli and for detection of the mutant b 5 during the purification procedure.…”

Uranyl Binding to Proteins and Structural-Functional Impacts

“…Moreover, both Th 4+ and UO 2 2+ ions reduce the oxy-form of Hb at a high concentration of >100 µM [40]. Cyt b 5 is a small membrane binding heme protein, and its heme-binding domain is highly negatively charged by a series of acidic residues, forming an "acidic" cluster [41][42][43]. Spectroscopic study showed that uranyl may bind to the surface of Cyt b 5 with a binding affinity of K D = 10 µM, and molecular modeling study suggested a possible uranyl binding site at surface residues, Glu37 and Glu43 (Figure 2a) [44,45].…”

The role of cytochrome b 5 structural domains in interaction with cytochromes P450

“…Expression vectors pCWori+ and pT7 were kind ly provided by Dr. R. W. Estabrook (UT Southwestern Medical Center, USA). For expression of cytochromes P450 and b 5 the following plasmids were used: pCWori+CYP17Horse [17], pCWori+CYP17Human [17], pCWori+CYP17gpig [17], pCWori+P4503A4 [12], pCWori+Mc b 5 rat [18], pCWori+b 5 Om rat [19], pT7 b 5 om2 [19], pT7 b 5 om3 [19], pT7 b 5 Om human [20] and pOR262P450red [21].…”

“…The purity of protein samples of cytochrome b 5 were analyzed by gel electrophoresis in a 15% polyacrylamide gel under denaturing conditions [22] on a Mini Protean II instrument (Bio Rad, USA). The concentration of the purified b 5 was determined from the absolute absorption spectrum of the oxidized form of the heme protein (ε 413 = 117 mM -1 ·cm -1 ) [23]. Titration of CYP3A4 by various forms of cytochrome b 5 .…”