NMR studies of putidaredoxin: associations of putidaredoxin with NADH-putidaredoxin reductase and cytochrome p450cam.

ABSTRACT

To characterize the electron-transfer reaction in the P450cam monooxygenation system, the binding regions of putidaredoxin (Pdx) to NADH-putidaredoxin reductase (PdR) and P450cam were investigated using isotope-filtered NMR experiments in which uniformly 15N-labeled Pdx ([U-15N]Pdx) is mixed with unlabeled PdR and P450cam. By addition of PdR to Pdx, site specific signal broadening was observed for the N-H correlation peaks from Val-28, Glu-72, Ile-88, and Gln-105. Although previous studies have suggested the contribution from acidic amino acid residues on the G-helix of Pdx to the binding with PdR, no site specific broadening was observed for the resonances from these residues except for Glu-72. The lesser contribution of electrostatic interactions to the Pdx/PdR complex formation was also suggested by our previous study (M. Aoki, K. Ishimori, H. Fukada, K. Takahashi, I. Morishima, Biochim. Biophys. Acta 1384 (1998) 180-188), which is in sharp contrast to the complex formation between adrenodoxin and adrenodoxin reductase. Upon the complex formation between Pdx and P450cam, the site specific NMR line broadening was observed for several amino acid residues distributed near the iron-sulfur cluster, corresponding to the large binding site in the complex formation with P450cam. Since some of the amino acid residues included in the binding site are not conserved for the electron-transfer iron-sulfur proteins such as ferredoxin and adrenodoxin, the interactions formed by these amino acid residues would be highly specific to the binding with P450cam, consistent with very low cross-reactivity to other iron-sulfur proteins in the P450cam monooxygenation system.