Abstract
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 K(d) 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 ((73)SAGGKAAA(80)) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor ((73)SASGKQAA(80)). 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 Ser(75) against the 2'-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding.
Highlights
Cytochrome P450 (P450)1 consists of a group of hemoprotein enzymes that are involved in the metabolism of a variety of ʈ To whom correspondence should be addressed
We further showed that a poor electron donor (NADPH) could bind Fnor, but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser75 against the 2-phosphate moiety
These results indicate that position 75 must be Gly for NADPH to be an effective electron donor of Nitric oxide reductase (Nor) activity as observed in wild type Cnor2 or Tnor
Summary
Cytochrome P450 (P450) consists of a group of hemoprotein enzymes that are involved in the metabolism of a variety of ʈ To whom correspondence should be addressed. Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is involved in fungal denitrification [6] This enzyme is the most functionally diverse among known P450 enzymes as it catalyzes the reaction shown in Reaction 1 without a redox partner such as P450 reductase and receives electrons directly from NADH or NADPH (NAD(P)H). We have isolated and cloned the genes for several fungal P450nor isoforms that belong to the CYP55A subfamily (10 – 13) These are P450norA and P450norB from F. oxysporum (Fnor) [14], P450nor and P450nor from Cylindrocarpon tonkinense (Cnor and Cnor2) [12, 15], and P450nor from T. cutaneum (Tnor) [13]. The Fnor species has been extensively analyzed both physicochemically and kinetically, and the structure has been determined by x-ray crystallography [16] These P450nor isoforms are specific for the electron donors, NADH and NADPH. The P450 superfamily has attained vast diversity by rapidly changing the substrate recognition sites (SRSs) of its many
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