Thomas M. Shea

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20–40 residues longer in the C terminus, forming a “tail” that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7–21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.

Assay of isoforms of Escherichia coli-expressed nitric oxide synthase.

The techniques described herein have added to our repertoire of experimental approaches for the characterization of the NOSs. These procedures have reinforced our conviction that the NOSs are structurally suited to perform unique functions in their cellular milieux and that these differences have physiological consequences.

Crystallization studies of NADPH-cytochrome P450 reductase.

Publisher Summary This chapter focuses on the crystallization studies of NADPH-cytochrome P450 reductase (CPR). NADPH-cytochrome P450 reductase is an essential component of the microsomal cytochrome P450 monooxygenase system. It is an integral membrane protein that catalyzes the transfer of electrons from NADPH to cytochrome P450 in the oxidative metabolism of the endogenous and exogenous substrates. The three-dimensional structure of CPR by X-ray analysis can reveal, at the molecular level, the interactions among the pyridine nucleotide NADPH, the flavins, and the polypeptide chain of CPR, and the spatial relationship among the different domains of CPR that allow for efficient electron transfer. In addition, the nature of the binding site for cytochrome P450 can be identified, providing the structural basis for the ability of CPR to interact with scores of different cytochromes P450. Obtaining crystals of CPR establishes the basis for the full structural analysis of CPR by X-ray diffraction methods. The crystallization conditions obtained from the CPR studies can be used as a starting point for crystallization of other FMN- and FAD-containing proteins, such as nitric oxide synthase.