In contrast to P450 reductase, the role of cytochrome b5 as partner in P450-dependent reactions is considerably more complex. There is a single cytochrome b5 gene in insect genomes, although there are several genes with cyt b5 -like domains.

The house fly cytochrome b5 is a 134 amino acid protein (Guzov et al., 1996) with 48% sequence identity to the orthologous rat cytochrome b5. Its N-terminal domain of about 100 residues is the heme-binding domain that is about 60% identical that of the vertebrate cytochrome b5. Its C-terminal portion is a hydrophobic membrane anchor. The Drosophila ortholog (CG2140) is 76% identical to the house fly cytochrome b5. The Helicoverpa armigera cytochrome b5 (Ranasinghe and Hobbs, 1999a) is 127 aa in length and 51% identical to the house fly cytochrome b5, and the Anopheles gambiae cytochrome b5 is 54% identical (Nikou et al., 2003).

The house fly cytochrome b5 protein was produced in E. coli, purified and fully characterized (Guzov et al., 1996). Absorption spectroscopy and EPR revealed properties very similar to cytochromes b5 from vertebrates. NMR spectra indicated that the orientation of the heme in the protein relative to its α,γ meso axis is about 1:1. This means that the protein is present in two forms of approximately equal abundance, that result from two modes of insertion of the non-covalently bound heme in the protein between the two coordinating histidines (face up and face down).

The X-ray crystal structure of house fly cytochrome b5 was solved at 1.55 A (PDB 2IBJ), revealing a small hydrophobic patch around Met 71 that contributes to the high stability of the holoprotein (Wang et al., 2007).

A redox potential of -26 mV was measured by cyclic voltammetry on a treated gold electrode in the presence of hexamminechromium(III) chloride, and was verified by classical electrochemical titration. Stopped flow spectrophotometry showed that the cytochrome b5 is reduced by house fly P450 reductase at a high rate (5.5 s-1)(Guzov et al., 1996).

Cytochrome b5 can also be reduced by its own reductase, an NADH-dependent FAD flavoprotein (Zhao et al., 2012), and can therefore provide either NADH-or NADPH-derived electrons to P450 enzymes. NADH-cytochrome b5 reductase and cytochrome b5 are also known to provide electrons to other acceptors, such as fatty acid desaturases and elongases.

Depending on the P450 enzyme and on the reaction catalyzed, cytochrome b5 may be either stimulatory, inhibitory, without effect, or its presence may be obligatory. Cytochrome b5 can have a quantitative effect on overall reaction rates, and/or a qualitative role on the type of reaction catalyzed and the ratio of the reaction products.

Cytochrome b5 can also influence the overall stoichiometry of the P450 reaction, in particular the “coupling rate”, i.e. the utilization and fate of electrons from NADPH relative to monooxygenation. Cytochrome b5 should therefore be regarded as an important modulator of microsomal P450 systems. General reviews of the role of cytochrome b5 in P450 reactions are available (Porter, 2002, Schenkman and Jansson, 2003) and known examples of this modulator role in insect systems follow.

The relative contribution of NADH in P450 reactions, but more importantly the NADH synergism of NADPH-dependent reactions that is occasionally observed, is probably attributable to cytochrome b5 as redox partner. Indeed, the Km of the P450 reductase for NADH is a thousand-fold higher than for NADPH, and the Vmax 10-fold lower (Murataliev et al., 1999), so that the contribution of NADH under normal conditions is probably channeled by NADH-cytochrome b5 reductase and cytochrome b5.

An anti-cytochrome b5 antiserum severely inhibited (up to 90%) methoxycoumarin and ethoxycoumarin O-dealkylation and benzo[a]pyrene hydroxylation, but not methoxyresorufin and ethoxyresorufin O-dealkylation when assayed in microsomes of the house fly LPR strain (Zhang and Scott, 1994). This antiserum also inhibits cypermethrin 4'-hydroxylation by these CYP6D1-enriched microsomes (Zhang and Scott, 1996a).

House fly cytochrome b5 stimulates heptachlor epoxidation and steroid hydroxylation when reconstituted with cytochrome P450 reductase, house fly CYP6A1 and phospholipids (Guzov et al., 1996; Murataliev et al., 2008). Stimulation of cyclodiene epoxidation and diazinon metabolism were also observed with Drosophila CYP6A2 expressed with the baculovirus system (Dunkov et al., 1997). Cytochrome b5 is efficiently reduced by P450 reductase, but it does not increase the rate of P450 reduction by P450 reductase. Because of its small redox potential, cytochrome b5 is unlikely to play an important role in delivering the first electron to P450 catalysis, and its stimulatory role probably involves an increased rate of transfer of the second electron. Cytochrome b5 decreases the apparent Km for P450 reductase and increases the Vmax for epoxidation at constant CYP6A1 concentrations (Guzov et al., 1996). The results suggest a role for cytochrome b5 in the P450 reductase - P450 interactions.

Whereas heptachlor epoxidation by CYP6A1 was increased 2-3 fold by the addition of cytochrome b5, the hydroxylation of testosterone, androstenedione and progesterone was stimulated 7-10 fold. The addition of cytochrome b5 increased the ratio of 2β-hydroxylation over 15β-hydroxylation of testosterone. This suggests that cytochrome b5 can have an effect on CYP6A1 conformation, probably altering the interaction of the binding site with either C-17 hydroxyl group (decreased) or the C-3 carbonyl (increased). Interestingly, the effect of cytochrome b5 on hydroxylation regioselectivity was also obtained with apo-b5 (cytochrome b5 depleted of heme and therefore redox incompetent), whereas the effect on turnover number was only much smaller with apo-b5 (Murataliev et al., 2008). The effect of apo-b5 is not due to heme transfer from P450 to apo-b5, and in fact, both apo-b5 and (holo) cytochrome b5 were shown to stabilize the ferrous-CO complex of CYP6A1, decreasing the rate its conversion to P420 (Murataliev et al., 2008).

Cytochrome b5 increases the coupling stoichiometry of CYP6A1 catalysis:

From Murataliev et al., 2008, Table 3. Stoichiometries of heptachlor epoxidation by purified house fly CYP6A1, reconstituted with purified CPR and cyt-b5. Rates are in µM/min/µM CYP6A1 (+SD).

Cytochrome b5 also stimulates the CYP6CM1vQ - catalyzed dealkylation of several alkoxycoumarin and resorufin (Karunker et al., 2009), the metabolism of permethrin and deltamethrin by CYP6M2 (Stevenson et al., 2011) and the dealkylation of ethoxycoumarin by CYP6FD1 (Liu et al., 2020).

Cytochrome b5 enhances the activity of Anopheles funestus CYP6P9a and b towards 7-benzyloxymethoxy-4-trifluoromethylcoumarin, but does not increase deltamethrin metabolism (Nolden et al., 2022).

Coordinate induction and/or overexpression of cytochrome b5 and P450 genes has been reported (Liu and Scott, 1996, Kasai et al., 1998b, Ranasinghe and Hobbs, 1999a, 1999b, Nikou et al., 2003) and this indicates that the effects of cytochrome b5 seen in vitro are probably significant in vivo as well.