Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains:

The three-dimensional strcture of mediumchain acyl-CoA dehydrogenase from pig mitochondria in the native form and that ofa complex ofthe enzyme and a strate (product) have been solved and refined by x-ray crystallo- Mammalian acyl-CoA dehydrogenases [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyze the first step in each cycle of fatty acid a-oxidation in mitochondria (1).Acyl-CoA thioesters are oxidized to the corresponding trans-2,3-enoyl-CoA products with concomitant reduction of enzyme-bound FAD. Reoxidation of the dehydrogenase flavin and the transfer of reducing equivalents to the mitochondrial respiratory chain are catalyzed by the soluble electron transferring flavoprotein (ETF) and the particulate ETF-ubiquinone oxidoreductase, an iron-sulfur flavoprotein (2, 3).Three soluble stright-chain acyl-CoA dehydrogenases have been isolated and classified according to their distinct but overlapping substrate specificities for long-, medium-, and short-chain fatty acids (4). Recently, a very-long-chain acylCoA dehydrogenase has been identified in the inner membrane of rat mitochondria (5). In addition, three dehydrogenases involved in amino acid metabolism, isovaleryl-(6), 2-methyl branched chain (7), and glutaryl-(8) CoA dehydrogenases, have been isolated and characterized. With the exception of the membrane-associated very-long-chain acylCoA dehydrogenase, these enzymes appear very similar in their catalytic mechanism and their biochemical properties.They are homotetramers and each subunit contains --400 aaThe publication costs of this article were defrayed in part by page chare payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. residues and one equivalent of FAD. Medium-chain acylCoA dehydrogenase (MCAD) exhibits a broad chain-length specificity and has its highest activity with Cg-CoA. Re-cently, several genetic diseases have been found to be due to acyl-CoA dehydrogenase deficiencies. Deficiency of MCAD appears to be the most common among disorders offatty acid oxidation in humans (9). It is manifested by fasting intolerance, hypoglycemic coma, failure of ketogenesis, dicarboxylic acidemia, and sudden infant death (10). Although the enzyme structure described in this report is that of the porcine liver enzyme, the amino acid sequence is very similar to that of the human enzyme (11), from which it differs by <10% conservative substitutions (A. W. Strauss, personal communication). Therefore, it is expected that structural conclusions drawn from the pig enzyme will apply to the human enzyme. We have reported the crystal structure of pig-liver MCAD at 3.0-A resolution (12) and preliminary data on complexes of the enzyme and substrates (13). In this paper, we present the crystal structure ofMCAD in the native form and that of a complex of the enzyme and the octanoylCoA, both refined at 2.4-A resolution.

Nitric-oxide synthase (NOS) is composed of a C-termi- Nitric-oxide synthase (NOS)1 catalyzes the NADPH-dependent conversion of L-arginine to nitric oxide and L-citrulline (for reviews, see Refs. 1 and 2). Three isoforms of NOS have been identified in mammals; neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, and their activities are Ca 2ϩ /calmodulin-dependent, whereas inducible NOS (iNOS) is independent of intracellular Ca 2ϩ concentration. Although the three isoforms share 50 -60% sequence identity, they differ in size, intracellular location, and regulation. nNOS has a molecular mass of 165 kDa, is located in neurons in the brain and at neuromuscular junctions, and is involved in neurotransmission. eNOS has a molecular mass of 133 kDa, is present in vascular endothelial cells, and is involved in vascular homeostasis. iNOS has a molecular mass of 130 kDa, is located in macrophages, and is expressed only in response to endotoxin or inflammatory cytokines. A distinguishing feature of NOS is that the N-terminal half of the enzyme is the hemebinding domain (or oxygenase domain), similar to the cytochrome P450 enzyme family, and also contains pterin-and arginine-binding sites. The C-terminal half is the flavin-binding or reductase domain containing FAD-, FMN-, and NADPHbinding sites, as found in NADPH-cytochrome P450 oxidoreductase (CYPOR) (3). At the junction between these two domains is a calmodulin (CaM)-binding region. Due to the reversible binding of CaM, the constitutive isoforms (nNOS and eNOS) display Ca 2ϩ dependence in their enzymatic activities (4, 5), whereas iNOS has tightly bound CaM, almost independent of the Ca 2ϩ concentration (6). The reductase domain can be further divided into two subdomains, each of which contains one of the two flavins. The FMN-containing subdomain is situated at the N terminus, and its amino acid sequence is similar to that of flavodoxin, whereas the C-terminal portion contains FAD-and NADPH-binding sites and is homologous to ferredoxin NADP ϩ reductase (FNR). From the sequence alignments, Salerno et al. (7) showed additional sequences in the middle of the FMN subdomain of the constitutive NOS isoforms that were not present in iNOS, CYPOR, or flavodoxin. It was proposed that this additional sequence of about 40 residues acts as an autoinhibitory element in the constitutive NOS isoforms that is released upon binding of Ca 2ϩ /CaM, as seen in other Ca 2ϩ /CaM-activated proteins, such as myosin light chain kinase. Deletion of this insert in nNOS (8) and eNOS (9, 10) results in enzymes that require significantly lower Ca 2ϩ concentrations for optimal activity. In addition, removal of this insert greatly enhances the maximal activity of eNOS, which is the least active of the three isozymes. Another significant difference between NOS and CYPOR is that the NOS isozymes contain an additional 21-42-amino acid tail in the C terminus, not present in CYPOR and FNR, which shares about 42% sequence homology with the FAD/NADPHbinding domains of CYPOR and NOS. Roma...

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP ؉ has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the reface of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP ؉ lies against the FAD isoalloxazine ring with a tilt of ϳ30°b etween the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and -interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.NADPH-cytochrome P450 oxidoreductase (CYPOR, 1 EC 1.6.2.4) is a 78-kDa flavoprotein bound to the cytoplasmic surface of the endoplasmic reticulum and the outer membrane of the nuclear envelope in eukaryotic cells. It contains two flavin cofactors, one FAD and one FMN, and functions in the sequential transfer of two reducing equivalents from NADPH to the heme center of cytochromes P450 via FAD and FMN (for reviews, see Refs. 1 and 2). Other physiological electron acceptors of CYPOR include heme oxygenase (3), cytochrome b 5 (4), and fatty-acid elongase (5). Although non-physiological, CYPOR is also capable of reducing cytochrome c in vitro. Kinetic, spectroscopic, and potentiometric studies using a reconstituted liver microsomal monooxygenase system indicate that the hydride ion is transferred from NADPH to the lower redox potential flavin, FAD (6, 7). FAD then transfers single electrons to FMN, which in turn reduces the catalytic heme center of cytochrome P450 (6 -8). Other eukaryotic enzymes known to contain two different flavin molecules within the same peptide chain, and that bind pyridine nucleotides, are the nitric-oxide synthase isozymes (NOS) (9), methionine synthase reductase (10), and NR1 (11). This relationship is extended to sequence homology, where CYPOR shares 30 -40% identity with the reductase domains of the NOS isozymes, 38% with methionine synthase reductase, and 35% with NR1. Sequence analysis shows CY-POR to be divided into two domains, a flavodoxin-like FMN domain, located in the N-terminal half of the molecule...