Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes.

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 A resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin-NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 A. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

[1]  B. Horecker.  TRIPHOSPHOPYRIDINE NUCLEOTIDE-CYTOCHROME c REDUCTASE IN LIVER , 1950 .

[2]  B. Brodie,et al.  The oxidation of drugs by liver microsomes: on the role of TPNH and oxygen. , 1957, The Journal of pharmacology and experimental therapeutics.

[3]  H. Kamin,et al.  Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver. , 1962, The Journal of biological chemistry.

[4]  A. H. Phillips,et al.  Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. , 1962, The Journal of biological chemistry.

[5]  B. Masters,et al.  The mechanism of 1- and 2-electron transfers catalyzed by reduced triphosphopyridine nucleotide-cytochrome c reductase. , 1965, The Journal of biological chemistry.

[6]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[7]  C. Kasper.  Biochemical distinctions between the nuclear and microsomal membranes from rat hepatocytes. , 1971, The Journal of biological chemistry.

[8]  B. Masters,et al.  Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system. , 1972, The Journal of biological chemistry.

[9]  T. Iyanagi,et al.  Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase. , 1973, Biochemistry.

[10]  L. Sieker,et al.  The Binding of Riboflavin-5′-Phosphate in a Flavoprotein: Flavodoxin at 2.0-Å Resolution , 1973 .

[11]  T. Iyanagi,et al.  Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b5 reductases. , 1974, Biochemistry.

[12]  M. J. Coon,et al.  Identification of the high and low potential flavins of liver microsomal NADPH-cytochrome P-450 reductase. , 1978, The Journal of biological chemistry.

[13]  H. Enoch,et al.  Cytochrome b5 reduction by NADPH-cytochrome P-450 reductase. , 1979, The Journal of biological chemistry.

[14]  Y. Yasukochi,et al.  NADPH-cytochrome c (P-450) reductase. Spectrophotometric and stopped flow kinetic studies on the formation of reduced flavoprotein intermediates. , 1979, The Journal of biological chemistry.

[15]  David Eisenberg,et al.  Unbiased three-dimensional refinement of heavy-atom parameters by correlation of origin-removed Patterson functions , 1983 .

[16]  B. C. Wang.  Resolution of phase ambiguity in macromolecular crystallography. , 1985, Methods in enzymology.

[17]  Kurt Wüthrich,et al.  Secondary structure of the α-amylase polypeptide inhibitor Tendamistat from Streptomyces tendae determined in solution by 1H nuclear magnetic resonance , 1985 .

[18]  H. S. Mason,et al.  Flavins of NADPH-cytochrome P-450 reductase: evidence for structural alteration of flavins in their one-electron-reduced semiquinone states from resonance Raman spectroscopy. , 1985, Biochemistry.

[19]  C. Kasper,et al.  NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins. , 1986, Biochemistry.

[20]  Y. Nisimoto.  Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase. , 1986, The Journal of biological chemistry.

[21]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[22]  P. Karplus,et al.  Crystallographic analysis of the binding of NADPH, NADPH fragments, and NADPH analogues to glutathione reductase. , 1988, Biochemistry.

[23]  W. Backes,et al.  The effect of NADPH concentration on the reduction of cytochrome P-450 LM2. , 1988, Journal of Biological Chemistry.

[24]  T. E. Wilson,et al.  Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis. , 1989, The Journal of biological chemistry.

[25]  P. Bastiaens,et al.  Time-resolved fluorescence spectroscopy of NADPH-cytochrome P-450 reductase: demonstration of energy transfer between the two prosthetic groups. , 1989, Biochemistry.

[26]  P. Bonants,et al.  A 31P-nuclear-magnetic-resonance study of NADPH-cytochrome-P-450 reductase and of the Azotobacter flavodoxin/ferredoxin-NADP+ reductase complex. , 1990, European journal of biochemistry.

[27]  S. Snyder,et al.  Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase , 1991, Nature.

[28]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[29]  P. Karplus,et al.  Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. , 1991, Science.

[30]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[31]  C. Kasper,et al.  31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase. , 1992, Biochemistry.

[32]  C. C. Correll,et al.  Phthalate dioxygenase reductase: a modular structure for electron transfer from pyridine nucleotides to [2Fe-2S]. , 1992, Science.

[33]  J. Findlay,et al.  The haemoglobin‐like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C‐terminal domain shares homology with ferredoxin NADP+ reductases , 1992, FEBS letters.

[34]  M. Waterman,et al.  Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities. , 1994, The Journal of biological chemistry.

[35]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[36]  C. Wolf,et al.  Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[37]  K. Inaka,et al.  Crystal structure of NADH-cytochrome b5 reductase from pig liver at 2.4 A resolution. , 1995, Biochemistry.

[38]  B. Masters,et al.  Flavin-binding and protein structural integrity studies on NADPH-cytochrome P450 reductase are consistent with the presence of distinct domains. , 1995, Archives of biochemistry and biophysics.

[39]  P. Karplus,et al.  Refined crystal structure of spinach ferredoxin reductase at 1.7 A resolution: oxidized, reduced and 2'-phospho-5'-AMP bound states. , 1995, Journal of molecular biology.

[40]  C. Kasper,et al.  Role of Acidic Residues in the Interaction of NADPH-Cytochrome P450 Oxidoreductase with Cytochrome P450 and Cytochrome c(*) , 1995, The Journal of Biological Chemistry.

[41]  G. Schneider,et al.  Structural studies on corn nitrate reductase: refined structure of the cytochrome b reductase fragment at 2.5 A, its ADP complex and an active-site mutant and modeling of the cytochrome b domain. , 1995, Journal of molecular biology.

[42]  S. Djordjević,et al.  Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[43]  C. W. Fisher,et al.  The interaction of NADPH-P450 reductase with P450: an electrochemical study of the role of the flavin mononucleotide-binding domain. , 1996, Archives of biochemistry and biophysics.