Roles of valine-98 and glutamic acid-72 of putidaredoxin in the electron-transfer complexes with NADH-putidaredoxin reductase and P450cam

In the P450cam reaction cycle, the interactions between putidaredoxin (Pdx) and P450cam or NADH-putidaredoxin reductase (PdR) have been considered to be essential in the electron-transfer process. In this study, three mutant putidaredoxins were prepared to examine the hydrophobic and electrostatic interactions in the reaction complexes. Val-98, which is exposed to solvent and has been previously suggested to play a key role in hydrophobic interactions, was substituted by alanine (V98A) or threonine (V98T) to perturb the hydrophobicity at the 98 position. Another mutation site was Glu-72 which is located in the middle of the negative charge-rich region. This glutamate was altered to glutamine (E72Q) to neutralize one of the negative charges on the surface of Pdx. The electronic absorption and H-1 NMR spectra of oxidized and reduced forms of these mutants, and their redox potentials were similar to those of wild type Pdx, indicating that the environment of the Fe-2-S-2 cluster was not very seriously affected by these mutations. In cytochrome c reduction activity, however, the ionic strength dependence of E72Q mutant differs slightly from that of the wild type protein. The mutation at glutamine at the 72 position weakened the association to PAR, indicating that Glu-72 is one of the amino acid residues contributing to form the reaction complex for the electron-transfer between Pdx and PdR. Based on the NADH consumption activity of these mutants, the hydrophobic interactions at Val-98 are involved in binding to P450cam, while electrostatic interactions at Glu-72 are rather small to affect the reaction between Pdx and P450cam. These different effects of the mutations suggest that the interaction sites to P450cam on Pdx are not completely superimposed on that to PUR. (C) 1998 Elsevier Science S.A. All rights reserved.