Abstract
Adrenodoxin reductase and adrenodoxin have been shown (Chu, J.-W., and Kimura, T. (1973) J. Biol. Chem. 248, 5183-5187) to form a low dissociation constant, 1:1 complex when both proteins are in the oxidized form. We have found that when adrenodoxin: adrenodoxin reductase ratios are varied by increasing the adrenodoxin concentration, with adrenodoxin reductase held constant, an increasing rate of cytochrome c reduction, with NADPH as reductant, is seen up to a ratio of 1:1, indicating that cytochrome c reduction occurs via the protein-protein complex. Spectra observed during titration of this protein-protein complex with NADH were resolved into components by the linear programming method, using a computer program written in Fortran IV. Analysis of the data has shown that the flavoprotein is reduced prior to the iron sulfur protein, and that the midpoint oxidation-reduction potentials (pH 7.5) of the two proteins are -295 and -331 mV, respectively, when both are present in the complex. Complex formation does not alter the potential of adrenodoxin reductase, but changes that of adrenodoxin by -40 mV. Equilibrium constants derived from potential measurements show that the strength of the protein-protein interaction in the complex is unaltered by reduction of adrenodoxin reductase, but is decreased by about 1 kcal due to reduction of adrenodoxin. The low dissociation constants for both oxidized reduced forms of the adrenodoxin reductase-adrenodoxin complex indicate that the complex must remain associated throughout its catalytic cycle. Titration of the adrenodoxin reductase-adrenodoxin complex with the physiologic reductant, NADPH, was followed by EPR and visible spectra, and yielded an order of reduction of the components identical with that seen when NADH was used as reductant. Reduction of the protein-protein complex with NADPH yielded a ternary complex between NADP+, flavoprotein, and iron sulfur protein, with the two electrons located in a "charge transfer" complex between flavoprotein and pyridine nucleotide.
Highlights
Adrenodoxin reductase and adrenodoxin have been shown (Chu, J.-W., and Kimura, T. (1973) J
We have found that when adrenodoxin: adrenodoxin reductase ratios are varied by increasing the adrenodoxin concentration, with adrenodoxin reductase held constant, an increasing rate of cytochrome c reduction, with NADPH as reductant, is seen up to a ratio of l:l, indicating that cytochrome c reduction occurs via the protein.protein complex
Analysis of the data has shown that the flavoprotein is reduced prior to the iron sulfur protein, and that the midpoint oxidation-reduction potentials of the two proteins are -295 and -331 mV, respectively, when both are present in the complex
Summary
The low dissociation constants for both oxidized and reduced forms of the adrenodoxin reductase.adrenodoxin complex indicate that the complex must remain associated throughout its catalytic cycle. Electrons from NADPH to the natural acceptor, cytochrome P450, or to cytochrome c in a convenient assay system, thereby suggesting a linear model for electron transport to these acceptors [2,3,4,5,6,7,8] Dyes such as dichloroindophenol (DCIP)’ can be reduced by the flavoprotein alone but the rate is substantially increased by addition of the iron sulfur protein [8, 11]. Namics of protein-protein interaction in this system have been investigated, and demonstrate that at low ionic strength the complex remains tightly associated regardless of the reduction state of the flavoprotein or iron sulfur protein
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