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J. Biol. Chem., Vol. 277, Issue 37, 33842-33847, September 13, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/37/33842    most recent M203923200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zhang, L. Articles by Shoun, H. Search for Related Content PubMed PubMed Citation Articles by Zhang, L. Articles by Shoun, H. Social Bookmarking                      What's this?

The B' Helix Determines Cytochrome P450nor Specificity for the Electron Donors NADH and NADPH^*

Li Zhang^§, Takashi Kudo, Naoki Takaya, and Hirofumi Shoun^¶

From the  Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan and the ^¶ Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

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 (⁷³SAGGKAAA⁸⁰) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor (⁷³SASGKQAA⁸⁰). 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.

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