The flavoprotein subcomplex of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria: insights into the mechanisms of NADH oxidation and NAD+ reduction from protein film voltammetry.
Complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria contains 45 different subunits and nine redox cofactors. NADH is oxidized by a noncovalently bound flavin mononucleotide (FMN), then seven iron-sulfur clusters transfer the two electrons to quinone, and four protons are pumped across the inner mitochondrial membrane. Here, we use protein film voltammetry to investigate the mechanisms of NADH oxidation and NAD+ reduction in the simplest catalytically active subcomplex of complex I, the flavoprotein (Fp) subcomplex. The Fp subcomplex was prepared using chromatography and contained the 51 and 24 kDa subunits, the FMN, one [4Fe-4S] cluster, and one [2Fe-2S] cluster. The reduction potential of the FMN in the enzyme's active site is lower than that of free FMN (thus, the oxidized state of the FMN is most strongly bound) and close to the reduction potential of NAD+. Consequently, the catalytic transformation is reversible. Electrocatalytic NADH oxidation by subcomplex Fp can be explained by a model comprising substrate mass transport, the Michaelis-Menten equation, and interfacial electron transfer kinetics. The difference between the "catalytic" potential and the FMN potential suggests that the flavin is reoxidized before NAD+ is released or that intramolecular electron transfer from the flavin to the [4Fe-4S] cluster influences the catalytic rate. NAD+ reduction displays a marked activity maximum, below which the catalytic rate decreases sharply as the driving force increases. Two possible models reproduce the observed catalytic waveshapes: one describing an effect from reducing the proximal [2Fe-2S] cluster and the other the enhanced catalytic ability of the semiflavin state.
Barker
Chérise D
CD
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK.
Reda
Torsten
T
Hirst
Judy
J
D000818:Animals; D002417:Cattle; D004563:Electrochemistry / Q000379:methods; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000378:metabolism
D005420:Flavoproteins; D021122:Protein Subunits; D009243:NAD; D042967:Electron Transport Complex I