Association and Redox Properties of the Putidaredoxin Reductase - Oxidized Nicotinamide Adenine Dinucleotide Complex

Published

November 13, 2007

Author(s)

Vytautas Reipa, Marcia J. Holden, V L. Vilker

Abstract

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the oxidized flavin adenine dinucleotide (FAD/FADH2) redox center is reduced by two-electron transfer from nicotinamide adenine dinucleotide (NAD+/NADH), which transforms these reducing equivalents in two separate, one-electron transfer steps to the iron-sulfur protein putidaredoxin (Pdx). In this communication, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of the equilibria among the redox components in this initial electron transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation-reduction potential of PdR that had been carefully purified of all residual NAD+, E0? = -369 10 mV, which is more negative than previously reported, and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). Measurements were made as a function of NAD+ concentration to show that at the saturating concentrations of pyridine nucleotide that would typically be found in an intracellular environment, E0? = -268 10 mV, which restores thermodynamically favorable electron transfer from NADH, and to the ferredoxin redox partner, putidaredoxin (E0? = -240 mV). Differential spectrometry was used to measure binding of NAD+ to oxidized PdR to form the PdRox:NAD+ charge-transfer complex (KD = 333 30 M). These results are integrated with known structural and kinetic information on PdR, as well as on AdR and FNR, in support of a compulsory ordered pathway for the electron transfer process catalyzed by all three reductases.

Citation

Journal of Biological Chemistry

Volume

46

Issue

45

Pub Type

Journals

Keywords

FAD, NAD+, putidaredoxin reductase, redox potential, spectroelectrochemistry

Citation

Reipa, V. , Holden, M. and Vilker, V. (2007), Association and Redox Properties of the Putidaredoxin Reductase - Oxidized Nicotinamide Adenine Dinucleotide Complex, Journal of Biological Chemistry (Accessed April 26, 2024)