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...

SUMMARY Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal new functions for these proteins (termed MXPs) we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend new mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

The freely diffusible and moderately reactive free radical, nitric oxide (NO), 2 is a biological signal molecule in numerous physiological and pathophysiological processes (for reviews, see Refs. 1-4). Nitric-oxide synthases (NOSs) catalyze the NADPH-dependent conversion of L-arginine to NO and L-citrulline (for reviews, see Refs. 5-7). In mammals, three different isoforms have been identified. Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, and their activities are Ca 2ϩ /CaM-dependent, whereas the inducible NOS (iNOS) is independent of intracellular Ca 2ϩ concentration. These isoforms share ϳ55% sequence identity yet differ in their size, tissue distribution, and regulation. The 165-kDa nNOS is located in neurons in the brain and neuromuscular junctions and is involved in neurotransmission. eNOS has a molecular mass of 133 kDa, is located in vascular endothelial cells, and is involved in vascular homeostasis. iNOS can be found in macrophages and many other tissues, has a molecular mass of 130 kDa, and is expressed only in response to endotoxins or inflammatory cytokines.All three isoforms of NOS are modular, homodimeric hemoflavoproteins. The N-terminal half of each NOS isozyme is similar to the cytochrome P450 enzyme family and contains iron protoporphyrin IX (heme). It is referred to as the heme domain or the oxygenase domain. This latter domain also contains tetrahydrobiopterin-and arginine-binding sites. The C-terminal half of each isozyme is the flavin-binding domain (or reductase domain) and contains FAD-, FMN-, and NADPH-binding sites, much the same as in NADPH-cytochrome P450 oxidoreductase (CYPOR). These two domains are linked by a CaM-binding region (8). The constitutive isoforms (nNOS and eNOS) are Ca 2ϩ -dependent due to their reversible binding of CaM, providing a mechanism for rapid response in a signaling cascade. On the other hand, iNOS has tightly bound Ca 2ϩ /CaM and is virtually independent of Ca 2ϩ concentration (9). In contrast to the other NOS isozymes, it is regulated at the transcriptional level. As in the case of the P450 (CYP)-CYPOR system, the FAD in the reductase domain accepts a pair of electrons in the form of a hydride ion from NADPH and transfers them one at a time to FMN. FMN, in turn, transfers the electrons again one by one to the heme of the other monomer in the NOS dimer (10 -12). However, the mechanisms of electron transfer and regulation of the FMN domain interactions with its electron acceptor (the heme domain) in NOS and related enzymes, including CYPOR and methionine synthase reductase, are largely unknown. Only recently, studies on this subject have been emerging (13)(14)(15)(16).CaM regulates a wide range of cellular functions through its reversible Ca 2ϩ -dependent binding to target proteins, including NOS. CaM regulates NOS activity by controlling the rates of electron transfer between the two flavin cofactors and between * This work was supported, in whole or in part, by National Institutes of Health Grant GM52682 (to J. J. K.

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.

NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85–90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR’s role with these multiple redox partners renders it a model for understanding protein–protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.