Abstract
Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in Escherichia coli complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213GH variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed.
Highlights
The universal cellular energy currency adenosine triphosphate (ATP) is mainly produced by oxidative phosphorylation, a process that couples electron transfer with ATP synthesis
A stable and homogeneous preparation of the complex produced from the nuo-operon on the plasmid is obtained in the presence of the detergent lauryl maltose neopentyl glycol (LMNG) by affinity- and size-exclusion-chromatography resulting in sufficient material of excellent quality for spectroscopic analysis (Figure 1)
To address the question whether the UV-vis redox-difference spectrum of the novel preparation of the E. coli complex I still provides an indication for a component that is neither the flavin mononucleotide (FMN) nor an Fe/S cluster, we repeated the experiments following the re-oxidation of a preparation that was reduced with NADH
Summary
The universal cellular energy currency adenosine triphosphate (ATP) is mainly produced by oxidative phosphorylation, a process that couples electron transfer with ATP synthesis. The released energy is used in an endergonic reaction to pump protons across the membrane, establishing the protonmotive force (pmf). Complex I contributes to the pmf by coupling NADH oxidation and quinone (Q) reduction with the translocation of protons across the membrane. Mammalian complex I is a complicated machinery consisting of up to 45 subunits, while bacteria contain a simpler version comprising in general 14 subunits.
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