Received June 19, 2007

Revised Manuscript Received September 4, 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 flavin adenine dinucleotide (FAD/FADH₂) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD⁺/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP⁺ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of 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 at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD⁺ (-330 mV). After addition of NAD⁺, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdR^ox:NAD⁺ + 2e^- PdR^rd:NAD⁺, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD⁺ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E⁰' = -230 ± 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH-cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD⁺ binding to oxidized PdR to establish that the PdR^ox:NAD⁺ complex (K_D = 230 M) is about 5 orders of magnitude weaker than PdR^rd:NAD⁺ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases.

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