The action of cytochrome b(5) on CYP2E1 and CYP2C19 activities requires anionic residues D58 and D65.

Cytochrome b5 (b5) is a small acidic protein with pleiotropic functions in human physiology, including hematology, endocrinology and metabolism, and xenobiotic metabolism. Encoded by a single CYB5A gene, a soluble form of b5 is found in erythrocytes, while a membrane-bound form is found in the endoplasmic reticulum in many tissues such as liver (1). The b5 protein has a heme prosthetic group with two histidine nitrogen atoms stably coordinating the heme iron, and b5 functions as a physiological one-electron transfer component of several reactions, including methemoglobin reduction, elongation and desaturation of fatty acids, and cholesterol biosynthesis (2). In addition, Hildebrandt and Estabrook first suggested in 1971 that b5 participates in cytochrome P450 (P450) reactions (3), and b5 thus stimulates androgen synthesis and regulates xenobiotic catabolism. Several lines of evidence support the physiologic importance of b5 in these various biochemical reactions. Genetic defects in b5 and more commonly in b5 reductase cause congenital methemoglobinemia (4, 5). The few carefully characterized male patients with inactivating CYB5A mutations bear a disorder of male sex development (46,XY DSD) with defective testosterone synthesis, specifically due to loss of CYP17A1-mediated 17,20-lyase activity and thus 19-carbon steroid synthesis (6–8). Furthermore, b5 is expressed in only one zone of the human adrenal gland, the zona reticularis, which is the major site of 19-carbon steroid synthesis in the human adrenal (9, 10). Finally, mice with liver-specific deletion of b5 show impaired metabolism of several drugs (11), which demonstrates that the capacity of b5 to stimulate P450-catalyzed oxidations is not an artifact of in vitro biochemical assays. Whereas the essential function of b5 in these biochemical systems is firmly established, the mechanism of its action in P450-catalyzed reactions remains unresolved and controversial. Depending on the specific P450 and substrate, b5 can be either an obligatory component or a modulator of reactions, which would occur in the absence of b5 (2, 12). Some P450s, including CYP2E1 and CYP2D6, are stimulated by b5 but not by b5 devoid of heme (apo-b5), suggesting that b5 functions as an electron transfer protein in these reactions. In contrast, other reactions, including those catalyzed by CYP3A4, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A5, CYP4A7, and CYP17A1, show equivalent stimulation in vitro upon addition of either b5 or apo-b5, suggesting that b5 might allosterically activate some P450s rather than directly participating in electron transfer (13–16). The heterogeneity amongst assay systems employed in these studies, the inconsistent results, and the potential for artifacts from heme transfer reactions (17) cloud our understanding of b5 action on P450 reactions and preclude the genesis of a unified model to explain these effects. The spectral changes induced upon b5 binding to various P450s have been employed to probe this interaction, but these differences are often small, slow, and difficult to reproduce (18), which limits the interpretations derived from these experiments (19). After many years of study, four different mechanisms have been proposed for the action of b5 in P450-mediated catalysis (2, 20). These mechanisms propose that 1) b5 directly transfers the second electron in the rate-limiting step of the P450 reaction; 2) b5 enhances the coupling between P450 and POR for product formation; 3) b5 accepts and holds an electron transferred first from POR to the P450, which facilitates rapid and consecutive transfer of two electrons to the P450 in a heterotrimeric complex, the first from POR and following molecular oxygen binding to the P450, the second from b5; and 4) b5 acts as an allosteric effector, which interacts with the P450•POR complex to stimulate catalysis without direct electron transfer. These models all invoke binary or ternary complex formation, which requires specific binding sites on b5 for the P450 and/or POR. Consequently, several studies have endeavored to identify the specific P450 residues responsible for interactions with b5. The CYP2B4 mutations R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all located on the proximal surface of the P450 near the heme ligand, exhibit attenuated affinity for b5 and reduced b5-stimulated methoxyflurane dehalogenation (21). More recently, mass spectrometry studies identified two intermolecular cross-links, between K428 and K434 of CYP2E1 with D53 and D56 of b5 (22), respectively, which provided the first direct experimental evidence for the interacting residues of a microsomal P450 and b5. Mutations R347H and R358Q in CYP17A1 cause isolated 17,20-lyase deficiency (23), and these mutations impair CYP17A1 interactions with both POR and b5 (24). In contrast to these studies defining key P450 residues, little attention has been directed to determining the critical residues of b5 required for P450 modulation. Our laboratory (25) established that E48 and E49 in human b5 are essential for stimulating 17,20 lyase activity of CYP17A1. The b5 mutation E48G+E49G retained only trace capacity to stimulate the 17,20-lyase reaction of CYP17A1 yet did not impair stimulation by wild-type b5 or lose capacity to bind heme and to accept an electron from POR. These results suggest that an allosteric interaction driven by electrostatic forces dominates the action of b5 on the CYP17A1•POR complex, which is consistent with the equivalence of b5 and apo-b5 in previous experiments (26). Given that apo-b5 does not stimulate CYP2E1-mediated oxidations and that mass spectrometry data suggest critical b5 residues other than E48 and E49 for these reactions, we probed the action of b5 on CYP2E1. We used a panel of b5 point mutations and targeted double mutations in reconstituted assays with POR and phospholipid to identify functionally important residues on b5 required for CYP2E1-catalyzed chlorzoxazone 6-hydroxylation. For comparison, we also studied the influence of these b5 mutations on the 17,20-lyase reaction of CYP17A1 and CYP2C19-catalyzed (S)-mephenytoin 4-hydroxylation.