M. Aoki et al., NMR-STUDIES OF PUTIDAREDOXIN - ASSOCIATIONS OF PUTIDAREDOXIN WITH NADH-PUTIDAREDOXIN REDUCTASE AND CYTOCHROME P450CAM, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1386(1), 1998, pp. 168-178
To characterize the electron-transfer reaction in the P450cam monooxyg
enation system, the binding regions of putidaredoxin (Pdx) to NADH-put
idaredoxin reductase (PdR) and P450cam were investigated using isotope
-filtered NMR experiments in which uniformly N-15-labeled Pdx ([U-N-15
]Pdx) is mixed with unlabeled PdR and P450cam. By addition of PdR to P
dx, site specific signal broadening was observed for the N-H correlati
on peaks from Val-28, Glu-72, Ile-88, and Gln-105. Although previous s
tudies have suggested the contribution from acidic amino acid residues
on the G-helix of Pdx to the binding with PdR, no site specific broad
ening was observed for the resonances from these residues except for G
lu-72. The lesser contribution of electrostatic interactions to the Pd
x/PdR complex formation was also suggested by our previous study (M. A
oki, K. Ishimori, H. Fukada, K. Takahashi, I. Morishima, Biochim. Biop
hys. Acta 1384 (1998) 180-188), which is in sharp contrast to the comp
lex 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 ne
ar the iron-sulfur cluster, corresponding to the large binding site in
the complex formation with P450cam. Since some of the amino acid resi
dues included in the binding site are not conserved for the electron-t
ransfer iron-sulfur proteins such as ferredoxin and adrenodoxin, the i
nteractions formed by these amino acid residues would be highly specif
ic to the binding with P450cam, consistent with very low cross-reactiv
ity to other iron-sulfur proteins in the P450cam monooxygenation syste
m. (C) 1998 Elsevier Science B.V. All rights reserved.