NMR-STUDIES OF PUTIDAREDOXIN - ASSOCIATIONS OF PUTIDAREDOXIN WITH NADH-PUTIDAREDOXIN REDUCTASE AND CYTOCHROME P450CAM

Citation
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
Citations number
25
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,Biophysics
ISSN journal
0167-4838
Volume
1386
Issue
1
Year of publication
1998
Pages
168 - 178
Database
ISI
SICI code
0167-4838(1998)1386:1<168:NOP-AO>2.0.ZU;2-7
Abstract
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.