title: Real-time electron transfer in respiratory complex I.
abstract: Electron transfer in complex I from Escherichia coli was investigated by an ultrafast freeze-quench approach. The reaction of complex I with NADH was stopped in the time domain from 90 mus to 8 ms and analyzed by electron paramagnetic resonance (EPR) spectroscopy at low temperatures. The data show that after binding of the first molecule of NADH, two electrons move via the FMN cofactor to the iron-sulfur (Fe/S) centers N1a and N2 with an apparent time constant of approximately 90 mus, implying that these two centers should have the highest redox potential in the enzyme. The rate of reduction of center N2 (the last center in the electron transfer sequence) is close to that predicted by electron transfer theory, which argues for the absence of coupled proton transfer or conformational changes during electron transfer from FMN to N2. After fast reduction of N1a and N2, we observe a slow, approximately 1-ms component of reduction of other Fe/S clusters. Because all elementary electron transfer rates between clusters are several orders of magnitude higher than this observed rate, we conclude that the millisecond component is limited by a single process corresponding to dissociation of the oxidized NAD(+) molecule from its binding site, where it prevents entry of the next NADH molecule. Despite the presence of approximately one ubiquinone per enzyme molecule, no transient semiquinone formation was observed, which has mechanistic implications, suggesting a high thermodynamic barrier for ubiquinone reduction to the semiquinone radical. Possible consequences of these findings for the proton translocation mechanism are discussed.
authors.affiliation: Helsinki Bioenergetics Group, Structural Biology and Biophysics Program, Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland. michael.verkhovsky@helsinki.fi
authors.forename: Marina L
authors.initials: ML
authors.lastname: Verkhovskaya
authors.forename: Nikolai
authors.initials: N
authors.lastname: Belevich
authors.forename: Liliya
authors.initials: L
authors.lastname: Euro
authors.forename: Mårten
authors.initials: M
authors.lastname: Wikström
authors.forename: Michael I
authors.initials: MI
authors.lastname: Verkhovsky
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D007700:Kinetics; D009243:NAD; D010084:Oxidation-Reduction; D016963:Thermus thermophilus
chemical_list: D009243:NAD; D042967:Electron Transport Complex I