Tags

Type your tag names separated by a space and hit enter

Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex.
Biochemistry. 2007 Nov 13; 46(45):13235-44.B

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, PdRox:NAD+ + 2e- --> PdRrd: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 PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd: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.

Authors+Show Affiliations

National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

17941648

Citation

Reipa, Vytas, et al. "Association and Redox Properties of the Putidaredoxin Reductase-nicotinamide Adenine Dinucleotide Complex." Biochemistry, vol. 46, no. 45, 2007, pp. 13235-44.
Reipa V, Holden MJ, Vilker VL. Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex. Biochemistry. 2007;46(45):13235-44.
Reipa, V., Holden, M. J., & Vilker, V. L. (2007). Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex. Biochemistry, 46(45), 13235-44.
Reipa V, Holden MJ, Vilker VL. Association and Redox Properties of the Putidaredoxin Reductase-nicotinamide Adenine Dinucleotide Complex. Biochemistry. 2007 Nov 13;46(45):13235-44. PubMed PMID: 17941648.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex. AU - Reipa,Vytas, AU - Holden,Marcia J, AU - Vilker,Vincent L, Y1 - 2007/10/17/ PY - 2007/10/19/pubmed PY - 2007/12/28/medline PY - 2007/10/19/entrez SP - 13235 EP - 44 JF - Biochemistry JO - Biochemistry VL - 46 IS - 45 N2 - 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, PdRox:NAD+ + 2e- --> PdRrd: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 PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd: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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/17941648/Association_and_redox_properties_of_the_putidaredoxin_reductase_nicotinamide_adenine_dinucleotide_complex_ L2 - https://doi.org/10.1021/bi7012118 DB - PRIME DP - Unbound Medicine ER -