Real-time optical studies of respiratory Complex I turnover
- Belevich, Nikolai
- Belevich, Galina
- Verkhovskaya, Marina
DOIAbstract
AbstractReduction of Complex I (NADH:ubiquinone oxidoreductase I) from Escherichia coli by NADH was investigated optically by means of an ultrafast stopped-flow approach. A locally designed microfluidic stopped-flow apparatus with a low volume (0.2μl) but a long optical path (10mm) cuvette allowed measurements in the time range from 270μs to seconds. The data acquisition system collected spectra in the visible range every 50μs. Analysis of the obtained time-resolved spectral changes upon the reaction of Complex I with NADH revealed three kinetic components with characteristic times of <270μs, 0.45–0.9ms and 3–6ms, reflecting reduction of different FeS clusters and FMN. The rate of the major (τ=0.45–0.9ms) component was slower than predicted...
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