A positively charged cluster formed in the heme-distal pocket of cytochrome P450nor is essential for interaction with NADH

Arg and Lys residues are concentrated on the distal side of cytochrome P450 nor (P450nor) to form a positively charged cluster facing from the outside to the inside of the distal heme pocket. We constructed mutant proteins in which the Arg and Lys residues were replaced with Glu, Gin, or Ala. The res ults showed that this cluster plays crucial roles in NADH interaction. We a lso showed that some anions such as bromide (Br-) perturbed the heme enviro nment along with the reduction step in P450nor-catalyzed reactions, which w as similar to the effects caused by the mutations. We determined by x-ray c rystallography that a Br-, termed an anion hole, occupies a key region neig hboring heme, which is the terminus of the positively charged cluster and t he terminus of the hydrogen bond network that acts as a proton delivery sys tem, A comparison of the predicted mechanisms between the perturbations cau sed by Br- and the mutations suggested that Arg(174) and Arg(64) play a cru cial role in binding NADH to the protein, These results indicated that the positively charged cluster is the unique structure of P450nor that responds to direct interaction with NADH.