Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is unique because it is catalytically self-sufficient, receiving electrons directly from NADH or NADPH. However, little is known about the direct binding of NADH to cytochrome. Here we report that oxidized pyridine nucleotides (NAD+ and NADP+) and an analogue induce a spectral perturbation in bound heme when mixed with P450nor. The P450nor isoforms are classified according to electron donor specificity for NADH or NADPH. One type (Fnor; a P450nor of Fusarium oxysporum) utilizes only NADH. We found that NAD+ induced a type I spectral change in Fnor whereas NADP+ induced a reverse type I spectral change, although the Kd values for both were comparable. In contrast, NADP+ as well as NAD+ caused a type I spectral change in Tnor, a P450nor isozyme from Trichosporon cutaneum that utilizes both NADH and NADPH as electron donors. The B’-helix region of Tnor (S73AGGKAAA80) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor (S73ASGKQAA80). A single mutation (S75G) significantly improved the NADPH-dependent Nor activity of Fnor, and the overall activity was accelerated via the NADPH-enhanced reduction step. These results showed that pyridine nucleotide cofactors can bind P450nor and that only a few residues in the B’-helix region determine cofactor specificity. We further showed that a poor electron donor (NADPH) could also bind Fnor but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser75 against the 2’-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding.