Conformational changes of NADPH-cytochrome P450 oxidoreductase are essential for catalysis and cofactor binding.

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. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.

The PPCD1 mouse: characterization of a mouse model for posterior polymorphous corneal dystrophy and identification of a candidate gene.

The PPCD1 mouse, a spontaneous mutant that arose in our mouse colony, is characterized by an enlarged anterior chamber resulting from metaplasia of the corneal endothelium and blockage of the iridocorneal angle by epithelialized corneal endothelial cells. The presence of stratified multilayered corneal endothelial cells with abnormal patterns of cytokeratin expression are remarkably similar to those observed in human posterior polymorphous corneal dystrophy (PPCD) and the sporadic condition, iridocorneal endothelial syndrome. Affected eyes exhibit epithelialized corneal endothelial cells, with inappropriate cytokeratin expression and proliferation over the iridocorneal angle and posterior cornea. We have termed this the "mouse PPCD1" phenotype and mapped the mouse locus for this phenotype, designated "Ppcd1", to a 6.1 Mbp interval on Chromosome 2, which is syntenic to the human Chromosome 20 PPCD1 interval. Inheritance of the mouse PPCD1 phenotype is autosomal dominant, with complete penetrance on the sensitive DBA/2J background and decreased penetrance on the C57BL/6J background. Comparative genome hybridization has identified a hemizygous 78 Kbp duplication in the mapped interval. The endpoints of the duplication are located in positions that disrupt the genes Csrp2bp and 6330439K17Rik and lead to duplication of the pseudogene LOC100043552. Quantitative reverse transcriptase-PCR indicates that expression levels of Csrp2bp and 6330439K17Rik are decreased in eyes of PPCD1 mice. Based on the observations of decreased gene expression levels, association with ZEB1-related pathways, and the report of corneal opacities in Csrp2bp(tm1a(KOMP)Wtsi) heterozygotes and embryonic lethality in nulls, we postulate that duplication of the 78 Kbp segment leading to haploinsufficiency of Csrp2bp is responsible for the mouse PPCD1 phenotype. Similarly, CSRP2BP haploinsufficiency may lead to human PPCD.

Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.

Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-cytochrome P450 oxidoreductase.

The microsomal flavoprotein NADPH-cytochrome P450 oxidoreductase (CYPOR) is believed to function as the primary, if not sole, electron donor for the microsomal cytochrome P450 mixed-function oxidase system. Development of the mammalian embryo is dependent upon temporally and spatially regulated expression of signaling factors, many of which are synthesized and/or degraded via the cytochromes P450 and other pathways involving NADPH-cytochrome P450 oxidoreductase as the electron donor. Expression of CYPOR as early as the two-cell stage of embryonic development (The Institute for Genomic Research Mouse Gene Index, version 5.0, www.tigr.org/tdb/mgi) suggests that CYPOR is essential for normal cellular functions and/or early embryogenesis. Targeted deletion of the translation start site and membrane-binding domain of CYPOR abolished microsomal CYPOR expression and led to production of a truncated, 66-kDa protein localized to the cytoplasm. Although early embryogenesis was not affected, a variety of embryonic defects was observable by day 10.5 of gestation, leading to lethality by day 13.5. Furthermore, a deficiency of heterozygotes was observed in 2-week-old mice as well as late gestational age embryos, suggesting that loss of one CYPOR allele produced some embryonic lethality. CYPOR -/- embryos displayed a marked friability, consistent with defects in cell adhesion. Ninety percent of CYPOR -/- embryos isolated at days 10.5 or 11.5 of gestation could be classified as either Type I, characterized by grossly normal somite formation but having neural tube, cardiac, eye, and limb abnormalities, or Type II, characterized by a generalized retardation of development after approximately day 8.5 of gestation. No CYPOR -/- embryos were observed after day 13.5 of gestation. These studies demonstrate that loss of microsomal CYPOR does not block early embryonic development but is essential for progression past mid-gestation.