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|Author(s):||Vytautas Reipa; Marcia J. Holden; V L. Vilker;|
|Title:||Association and Redox Properties of the Putidaredoxin Reductase - Oxidized Nicotinamide Adenine Dinucleotide Complex|
|Published:||November 13, 2007|
|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|
|Pages:||pp. 13235 - 13244|
|Keywords:||FAD,NAD+,putidaredoxin reductase,redox potential,spectroelectrochemistry|