The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Abstract

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b(5). In both instances, product formation occurs. When the second electron is donated by cytochrome b(5), catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b(5) has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b(5) on cytochrome P450 2B4 reactivity can explain how cytochrome b(5) is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b(5) to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b(5) to cytochrome P450 reductase, cytochrome b(5) inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b(5) are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b(5) stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.

Figure 1

Catalytic Cycle of Cytochrome P450

Figure 2

The binding site for cyt b5 and cyt P450 reductase on the proximal surface of cyt P450 2B4. The largely buried heme is in red; residues R422 and 443, involved in binding only the reductase, are illustrated in green; residues which participate in binding both cyt b5 and reductase in and near the C-helix (R122-K139) and K433 are shown in yellow. The figure was generated using the Midas Plus software system from the Computer Graphics Laboratory, University of California, San Francisco, [53] (Reprinted with permission of Elsevier, Biochem Biophys Res Commun 338 (2005) 499-506)

Figure 3

Cytochrome P450 2B4-cytochrome b₅ model complex bound to a membrane. Residues V66 and D65 of cytochrome b₅ are in contact with R126, K433, R122 of cytochrome P450 2B4. (PDB code of 1SUO).

Figure 4

Cytochrome P450-cytochrome b₅ model complex. The complex was generated with pymol and accounts for the available mutagenesis data. PDB code of P450 2B4 1SUO.

Figure 5

Cytochrome P450-reductase model complex bound to a membrane. The reductase is Molecule A (PDB Code 3ES9) of the open conformation of reductase.

Figure 6

Cytochrome P450-reductase model complex. The complex was generated with pymol and accounts for available mutagenesis data. PDB code of P450 2B4 1SUO and Molecule A PDB code 3ES9.

Figure 7

Comparison of the kinetics of the decay of oxyferrous cytochrome P450 to the ferric species and of norbenzphetamine formation in the presence of substrate, benzphetamine. The ΔA_438nm represents the decay of the oxyferrous P450 2B4 to the ferric protein [. The data for product formation are from [28].

, product formation by P450-b5;	, product formation by P450-CPR;
, ΔA438nm for P450-b5;	, ΔA438nm for P450-CPR.