NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. NADPH-cytochrome P450 oxidoreductase (CYPOR)4 is a ϳ78-kDa, multidomain, microsomal diflavin protein that shuttles electrons from NADPH 3 FAD 3 FMN to members of the ubiquitous cytochrome P450 superfamily (1, 2). In humans, the cytochromes P450 (cyt P450) are one of the most important families of proteins involved in the biosynthesis and degradation of a vast number of endogenous compounds and the detoxification and biodegradation of most foreign compounds. CYPOR also donates electrons to heme oxygenase (3), cytochrome b 5 (4), and cytochrome c (5).The FAD receives a hydride anion from the obligate two electron donor NADPH and passes the electrons one at a time to FMN. The FMN then donates electrons to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts electrons at two different steps in its complex reaction cycle. Ferric cyt P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the second of the two electrons to form the peroxo (Fe ϩ3 OO) 2Ϫ cyt P450 intermediate (6). In vivo, CYPOR cycles between the one-and three-electron reduced forms (7,8). Although the one-electron reduced form is an air-stable, neutral blue semiquinone (FMN ox/sq , Ϫ110 mV), it is the FMN hydroquinone (FMN sq/hq , Ϫ270 mV), not the semiquinone, that donates an electron to its redox partners (8 -11). CYPOR is the prototype of the mammalian diflavin-containing enzyme family, which includes nitric-oxide synthase (12), methionine synthase reductase (13,14), and a novel reductase expressed in the cytoplasm of certain cancer cells (15). CYPOR is also a target for anticancer therapy, because it reductively activates anticancer prodrugs (16).CYPOR consists of an N-terminal single ␣-helical transmembrane anchor (ϳ6 kDa) responsible for its local...

Background: cytb 5 modulates catalysis performed by cytsP450, in vivo and in vitro. Results: The structure of full-length cytb 5 was solved by NMR, and the cytP450-binding site on cytb 5 was identified by mutagenesis and NMR. Conclusion: A model of the cytb 5 -cytP450 complex is presented. Addition of a substrate strengthens the cytb 5 -cytP450 interaction. Significance: The cytb 5 -cytP450 complex structure will help unravel the mechanism by which cytb 5 regulates catalysis by cytP450.

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp 147 and Arg 514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (>90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function.

1 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 b5. In both instances, product formation occurs. When the second electron is donated by cytochrome b5, 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 b5 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 b5 on cytochrome P450 2B4 reactivity can explain how cytochrome b5 is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b5 to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochome b5 to cytochrome P450 reductase, cytochrome b5 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 b5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b5 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.

The cytochromes (cyt) 2 P450 are a superfamily of heme-containing enzymes that catalyze the biotransformation of a large number of endogenous and xenobiotic compounds by utilizing reducing equivalents from NADPH. The mammalian hepatic microsomal cytochromes P450 receive electrons from their redox partner, NADPH-cyt P450 reductase (CPR). To be able to catalyze the oxidation of substrates, cyt P450 requires two electrons and two protons. Its catalytic cycle is complex with multiple steps (1). The first step is the binding of substrate, which may increase the redox potential of the heme. CPR, not cyt b 5 , then delivers the first electron to reduce the ferric heme to the ferrous state. The ferrous iron binds oxygen to form the oxyferrous complex (Fe 3ϩ OO Ϫ ), whose potential is high enough to accept a second electron from either CPR or cyt b 5 to yield the peroxo intermediate ( . A second proton is delivered to the distal oxygen, resulting in cleavage of the oxygen molecule to form water and the oxyferryl intermediate [Fe IV ϭ O], generally considered to be the active hydroxylating species. An atom of oxygen is inserted into the substrate, and product release follows.It has been recognized for 3 decades that under steady-state conditions, cyt b 5 alters the rate of catalysis by selected cytochromes P450 (for recent reviews see Refs. 2-5). Cyt b 5 has been reported to affect the catalytic activity of more than 20 cyt P450 isoforms, including the majority of the human drug-metabolizing cyt P450 isoforms like cyt P450 3A4, 2B6, 2C9, 2C19, and 2E1 (6 -9). Cyt b 5 may enhance, inhibit, or not alter catalysis by microsomal cyt P450 depending on the specific cyt P450 isoform, the substrate, and the experimental conditions (10, 11). It has been suggested that cyt b 5 modulates cyt P450 catalysis by donating the second electron to cyt P450 and/or by acting as an allosteric modifier of the oxygenase (3). In the case of cyt P450 2B4, it has been shown that the electron-donating properties of cyt b 5 are required for its stimulatory activity (10 -12). Its inhibitory properties, which are also manifest by manganese-protoporphyrin IX cyt b 5 , are likely related to its ability to displace CPR from its binding site on cyt P450 2B4 (11-13). Recent experiments performed under single turnover conditions suggest that both the electron-donating ability and effec-

Cytochrome b5 (cyt b5) is a membrane-anchored electron-carrier protein containing a heme in its soluble domain. It enhances the enzymatic turnover of selected members of the cytochrome P450 superfamily of catabolic enzymes, localized in the endoplasmic reticulum of liver cells. Remarkably, its alpha-helical membrane-anchoring domain is indispensable for the cyt b5/cyt P450 interaction. Here, we present the first solid-state NMR studies on holo-cyt b5 in a membrane environment, namely, macroscopically oriented DMPC:DHPC bicelles. We have presented approaches to selectively investigate different domains of the protein using spectral editing NMR techniques that utilize the unique motional properties of each domain. Two-dimensional 1H-15N HIMSELF spectra showed PISA-wheel patterns reporting on the structure and dynamics of the membrane anchor of the protein.

The oxy-ferrous complex of cytochrome P450 2B4 (2B4) has been prepared at − 40°C with and without bound substrate (butylated hydroxytoluene, BHT), and radiolytically oneelectron cryoreduced at 77K. EPR shows that in both cases the observed product of cryoreduction is the hydroperoxo-ferriheme species, indicating that the microsomal P450 contains an efficient distal-pocket proton-delivery network. In the absence of substrate, two distinct hydroperoxo-ferriheme signals are observed, reflecting the presence of two major conformational substates in the oxy-ferrous precursor. Only one species is observed when BHT, is bound, indicating a more ordered active site. BHT binding also changes the g-tensor components of the hydroperoxo-ferric 2B4 intermediate, indicating that the substrate modulates the properties of this intermediate. Step-annealing the cryoreduced ternary 2B4 complex at 175K and above causes the loss of hydroperoxo-ferric 2B4 and the parallel appearance of high-spin ferri-2B4; LC-MS/MS analysis shows that in this process BHT is quantitatively converted to two products, hydroxymethyl BHT (1) and 3-hydroxy-t-butyl BHT (2). This implies that the hydroperoxo-ferric 2B4 prepared by cryoreduction is catalytically active, and that the high-spin state observed after annealing contains an enzymebound product of BHT monooxygenation. The ratio of products generated during cryoreduction/annealing, 1/2 ~ 6.2/1, is significantly different from the ratio, 2.5/1, at ambient temperature, and product coupling is significantly greater. This suggests that substrate is held more rigidly relative to the oxidizing species at low temperature, and/or that dissociation of FeOOH is inhibited. As in experiments under ambient conditions, product formation is not observed in the inactive F429H 2B4 mutant.

Microsomal monoxygenase enzymes of the cytochrome-P450 family are found in all biological kingdoms, and play a central role in the breakdown of metabolic as well as xenobiotic, toxic and 70% of the drugs in clinical use. Full-length cytochrome-b5 has been shown to be important for the catalytic activity of cytochrome-P450. Despite the significance in understanding the interactions between these two membrane-associated proteins, only limited high-resolution structural information on the full-length cytochrome-P450 and the cytochromes-b5-P450 complex is available. Here, we report a structural study on a functional ~72-kDa cytochromes-b5-P450 complex embedded in magnetically-aligned bicelles without having to freeze the sample. Functional and solid-state NMR (Nuclear Magnetic Resonance) data reveal interactions between the proteins in fluid lamellar phase bilayers. In addition, our data infer that the backbone structure and geometry of the transmembrane domain of cytochrome-b5 is not significantly altered due to its interaction with cytochrome-P450, whereas the mobility of cytochrome-b5 is considerably reduced.