The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Abstract

Cytochrome b 5 (cyt b 5 ) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane ␣-helix at its carboxyl terminus. These two domains are connected by an ϳ15-amino acid linker domain, Ser 90 -Asp 104 , which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b 5 to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b 5 to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b 5 except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b 5 to interact with cyt P450 2B4. A model for the membranebound cyt b 5 -cyt P450 complex is presented.Microsomal cytochrome b 5 (cyt b 5 ) 1 is an amphipathic electron transfer hemoprotein located in the membrane of the endoplasmic reticulum. It provides electrons for a broad range of reactions, including fatty acid desaturation, cholesterol biosynthesis, and a variety of cytochrome P450-dependent oxidation and hydroxylation reactions (1, 2).Cytochrome P450 (cyt P450) is responsible for the oxidation of a large number of substrates. The overall stoichiometry of the reaction catalyzed by cyt P450 is shown in Reaction 1, where RH is the substrate (3).The cyt P450 catalytic reaction occurs via a reaction cycle that involves substrate binding, reduction of the ferric heme, O 2 binding, reduction of the oxyferrous heme (second electron transfer), substrate oxidation, and finally product dissociation (4). Both ferric and oxyferrous cyt P450 receive electrons from NADPH via its redox partner, NADPH-cytochrome P450 reductase (cyt P450 reductase), whereas cyt b 5 can donate the second electron. Because of the high redox potential of Х20 mV for cyt b 5 , the first electron required to reduce cyt P450 from the ferric to the ferrous state is always transferred from NADPH via cyt P450 reductase. However, as the redox potential of cyt P450 increases from ХϪ230 mV to ϩ50 mV on transition to the oxyferrous state, the second electron can be obtained from either cyt P450 reductase or cyt b 5 (5, 6).Depending on the substrate, isozyme of cyt P450 and the experimental conditions cyt b 5 can stimulate, inhibit, or have no effect on cyt P450 activity (2). Studies of the stoichiometry of the metabolism of benzphetamine and methoxyflurane by cyt P450 2B4 have shown that cyt b 5 increases the efficiency of catalysis by cyt P450 2B4 primarily by decreasing the formation of the side-product superoxide. However, cyt b...

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

“…2B). The inability of the soluble haem core to stimulate the lyase activity is in agreement with other reconstituted CYP-CPR-cytochrome b 5 studies, indicating that the membrane-anchoring region provides not only correct membrane-integrated topology of cytochrome b 5 for maximal interaction with CYP17 but also facilitates protein-protein association through charge-based pairings (Lamb et al 2001, Clarke et al 2004. Moreover, Mulrooney et al (2004) have recently provided evidence that the hydrophobic interactions of the membraneanchoring regions occur via non-specific interactions.…”

“…There have been relatively few studies on cytochrome b 5 mutagenesis regarding its influence on CYP catalysis (Chudaev et al 2001, Clarke et al 2004 and as yet there have been no reports on the effect of cytochrome b 5 mutagenesis on the steroidal activity of CYP17. Earlier work by Usanov's group (Usanov & Chashchin 1991) showed that substitution of the residue Glu 42 with Lys on cytochrome b 5 resulted in reduction of the cholesterol side-chain cleavage activity of CYP11 by almost 40%, suggesting that the anionic residue of cytochrome b 5 was important for electrostatic interaction with CYP11.…”

ROLE OF CYTOCHROMEB5IN MODULATING PEROXIDE-SUPPORTED CYP3A4 ACTIVITY: EVIDENCE FOR A CONFORMATIONAL TRANSITION AND CYTOCHROME P450 HETEROGENEITY

“…The changes in the profiles of regioisomer formation from phenyldiazene suggest that in S119W (BЈ-C loop), active site topology is altered upon b 5 -interaction (Yamaguchi et al, 2003). In this context it is important to note that some basic amino acid residues in the C and CЈ helix are thought to be involved in binding between P450 2B4 and b 5 (Bridges et al, 1998;Clarke et al, 2004). Likewise, the substitutions at Ile-301 and Ala-305 in the SRS-4 region, which is closest to the heme (Williams et al, 2004;Yano et al, 2004), with larger side chains may cause a structural transition leading to impaired b 5 -interaction.…”

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

“…Activity of cytP4502B4 in Solution and Bicelles, Methoxyflurane-The metabolism of the anesthetic, methoxyflurane (C 3 H 4 Cl 2 F 2 O), by cytP4502B4 was measured in a reconstituted aqueous system with and without cytb 5 (23). The activity of cytP450 was also measured in bicelles in the presence and absence of cytb 5 .…”

“…Additionally, although the interaction of cytb 5 with various membranes has been previously studied (20 -22), the structure of membrane-bound cytb 5 is lacking. However, only the full-length membrane-binding form of microsomal cytb 5 influences the enzymatic activity of cytP450 (23,24); truncated cytb 5 is only capable of electron transfer to water-soluble oxidative enzymes (e.g. cytochrome c and metmyoglobin) (25).…”