D88A mutant of cytochrome P450nor provides kinetic evidence for direct complex formation with electron donor NADH.
The haem-distal pocket of nitric oxide reductase cytochrome P450 contains many Arg and Lys residues that are clustered to form a putative access channel for NADH. Asp88 is the sole negatively charged amino acid in this positive charge cluster, and thus it would be interesting to know its functional role. Here we found the intriguing phenomenon that mutation at this site of P450nor (D88A or D88V) considerably decreased the overall nitric oxide reductase activity without blocking the reducing half reaction in which the ferric enzyme-NO complex is reduced with NADH to yield a specific intermediate (I). The results indicate that the catalytic turnover subsequent to the I formation was blocked by such mutation. This property of the mutants made it possible to perform kinetic analysis of the reduction step, which is impossible with the wild-type P450nor. These results are the first kinetic evidence for direct complex formation between P450nor and an electron donor (NADH or NADPH). The kinetic analysis also showed that the inhibition by chloride ions (Cl(-)) is competitive with respect to NAD(P)H, which highlights the importance of the binding site for Cl(-) (the anion hole) in the interaction with NAD(P)H. We also characterized another mutant (D393A) of P450nor. The results demonstrated that both Asp residues play important roles in the interaction with NADH, whereas the role of Asp88 is unique in that it must be essential for the release of NAD(+) rather than binding to NADH.
Umemura
Mariko
M
Institute of Applied Biochemistry, University of Tsukuba, Japan.
Su
Fei
F
Takaya
Naoki
N
Shiro
Yoshitsugu
Y
Shoun
Hirofumi
H
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002712:Chlorides / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D002712:Chlorides; D046911:Macromolecular Substances; D009243:NAD; D001224:Aspartic Acid; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C080610:nitric-oxide reductase (P450)