Oxidation-reduction properties of short-chain acyl-CoA dehydrogenase: effects of substrate analogs.
Redox potentials of short-chain acyl-CoA dehydrogenase from the anaerobe, Megasphaera elsdenii, have been determined by means of uv-visible spectroelectrochemistry in the presence of substrate analogs. During redox titrations in the presence of 2-azabutyryl-CoA, up to 85% anionic FAD semiquinone was stabilized with a molar absorbance at 387 nm of 19 mM-1 cm-1. Despite a slow reduction of short-chain acyl-CoA dehydrogenase by 2-azabutyryl-CoA (< 2% reduction/h), a dissociation constant of 0.7 microM was measured and redox potentials, E1(0') and E2(0'), of -0.07 and -0.17 V, respectively, were determined at pH 7.0 for the first and second electrons in reduction of the FAD of short-chain acyl-CoA dehydrogenase. The analog, 2-azoctanoyl-CoA, did not reduce short-chain acyl-CoA dehydrogenase, bound with a dissociation constant of 2 microM, stabilized up to 47% anionic FAD semiquinone, and gave values of -0.08 and -0.11 V for E1(0') and E2(0') at pH 6.9. In contrast to 2-aza-acyl-CoA, the thioethers, butyl-CoA, octyl-CoA, and allyl-CoA, and the thioester, acetyl-CoA, did not bind strongly (Kd > or = 50 microM) and caused no significant change in the redox properties of short-chain acyl-CoA dehydrogenase. The two-electron redox potential, Em, remained at -0.08 V at pH 7.0 and there was no stabilization of FAD semiquinone in the presence of these analogs. These results show that no single feature of substrate structure, the thioester carbonyl, the presence of 2,3-unsaturation, or a fatty alkyl chain of appropriate length, can account for the 0.06-V positive change in redox potential which is observed in the presence of the substrate couple, crotonyl-CoA/butyryl-CoA (M. T. Stankovich and S. Soltysik (1987) Biochemistry 26 2627-2632). As outlined above, 2-azabutyryl-CoA or 2-azaoctanoyl-CoA did cause marked changes in the redox properties of short-chain acyl-CoA dehydrogenase, but the preferential stabilization of FAD semiquinone and negative change in Em distinguish the effects of 2-azaacyl-CoA from those of the substrate couple.
Pace
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Department of Chemistry, University of Minnesota, Minneapolis 55455.
Stankovich
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D000214:Acyl Coenzyme A / Q000378:metabolism* / Q000494:pharmacology; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D001372:Aza Compounds / Q000378:metabolism* / Q000494:pharmacology; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D010084:Oxidation-Reduction; D011199:Potentiometry / Q000379:methods; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C089170:2-azabutyryl-coenzyme A; D000214:Acyl Coenzyme A; D001372:Aza Compounds; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase