Structure of CPR

The structure of CPR is significant because this membrane-bound protein mediates electron transfer from NADPH to all microsomal P450 enzymes. A soluble form of CPR was generated by limited protease digestion and crystallized. The structure consists of a N-terminal FMN domain linked to... 

The presence of two coenzyme-binding sites is unexpected since they cannot be inferred solely from the crystal structure of CPR. Kinetic studies with wild t) e and W676H CPR at different concentrations of NADPH have, however, provided further support for the existence of two sites The rate of flavin reduction in the isolated FAD domain and CPR increases as NADPH is decreased from molar excess to stoichiometric concentrations. At stoichiometric concentration, the second noncatalytic site is predominantly vacant and the partial inhibition on the rate of flavin reduction from the catalytic site is therefore relieved (Figure 4.9). Occupation of the noncatalytic site occurs at NADPH concentrations in excess of the enzyme concentration, and impairs NADP" " release from the catalytic site. This in turn partially inhibits flavin reduction, the rate of which is gated by NADP release. Preincubation of the enzyme with a stoichiometric amount of adenosine 2, 5 -diphosphate does not lead to inhibition of the flavin reduction rate. We infer that the binding of adenosine 2, 5 -diphosphate prevents NADPH from binding to the noncatalytic site. This observation also suggests that it is the nicotinamide-ribose-phosphate portion of NADPH bound at the second site that hinders NADP" release from the catalytic site. Clearly, these new... 

The crystal structure of NADPH-cytochrome P450 reductase (CPR), the common electron-transfer protein of Class 11 eukaryotic P450 systems, was reported in 1997. This was followed by the structures of adrenodoxin reductase (AdR) and adrenodoxin (Adx), the two electron-transfer proteins of the Class 1 mitochondrial P450 system. The crystal structure of a cross-linked AdR-Adx complex has also been reported. " Putidaredoxin reductase (PdR) and putidaredoxin (Pd) of the P450cam system have also been structurally characterized. ... 

Figure 7 The crystal structure of NADPH-cytochrome P450 reductase (CPR). The FAD and FMN domains are linked by a hinge domain...

Synthesis and structure of [Fe(CpR)(Tp)] have been reported,29 and the electronic and magnetic properties have been studied and compared to their homoleptic analogs [Fe(CpR)2] and [FefTph]. [Fe(Cp )(Tp)j displays a spin equilibrium (S = 0 S = 2) in solution, investigated by variable temperature NMR spectroscopy.30... 

Another Class II P-450 redox system that has been extensively studied is the cytosolic flavocytochrome P-450 BM3 from Bacillus megaterium. BM3 is the fusion of a soluble P-450 domain with CPR." " " The FAD of BM3 is reduced by NADPH the electrons are transferred to FMN and then finally to the substrate-bound P-450 domain." " BM3 is the fastest reported P-450 monooxygenase,with the rate of hydride transfer from NADPH to FAD and the rate of electron transfer from FMN to heme several-fold above the mammalian P-450 redox systems." The structure of the full-length protein has yet to be solved, but the structure of the FAD- and NADPH-binding domain has been determined." This domain closely resembles rat liver CPR and contains several conserved residues implicated in NADPH binding and flavin reduction. 

The cDNA and corresponding primary amino acid sequences of several CPRs including rat , rabbit- , and human were obtained by the mid-1980s, and the development of Escherichia coli expression systems paved the way for detailed molecular characterization of the polypeptide through site-directed mutagenesis. The three-dimensional structure of rat CPR was determined by X-ray crystallography in 1997 by Kim and coworkers -, providing the structural prototype for dual flavin oxidoreductases. 

Central to the theoretical models of cardiac or thoracic pumping are two biomedical aspects. These are the actions of valves and the source of the blood volume that is being displaced. In Figure 18.1, a much-simplified representation of the relevant structures in CPR is given, to support and lend clarity to the modeling strategies described later. 

Given that the general structure of the FMN-binding domain of eukaiyotic CPR enzymes is highly related to that of microbial flavodoxins, it is perhaps not surprising that bacterial flavodoxins have been shown to act as redox... 

It has been observed that molybdocenes form monomeric and dimeric complexes upon dissolution in water and the solution structures of the molybdocenes at neutral pH are inferred as the monomeric [(CpR)2Mo(OH2)(OH)]+ and the dimeric [(CpR)2Mo(p-OH)2Mo(CpR)2] . The equilibrium constants for the monomer-dimer equilibria (shown in 26) have been obtained from H NMR dilution studies. ... 

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