FT-IR spectroscopic characterization of NADH : ubiquinone oxidoreductase (complex I) from Escherichia coli: Oxidation of FeS cluster N2 is coupled with the protonation of an aspartate or glutamate side chains

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, i s the first energy-transducing complex of many respiratory chains. It coupl es the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to nine iron-sulfur (FeS) c lusters participate in the redox reaction. So far, complex I has been descr ibed mainly by means of EPR- and UV-vis spectroscopy. Here, we report for t he first time an infrared spectroscopic characterization of complex I. Elec trochemically induced FT-IR difference spectra of complex I from Escherichi a coli and of the NADH dehydrogenase fragment of this complex were obtained for critical potential steps. The spectral contributions of the FMN in bot h preparations were derived from a comparison using model compounds and tur ned out to be unexpectedly small. Furthermore, the FT-IR difference spectra reveal that the redox transitions of the FMN and of the FeS clusters induc e strong reorganizations of the polypeptide backbone. Additional signals in the spectra of complex I reflect contributions induced by the redox transi tion of the high-potential FeS cluster N2 which is not present in the NADH dehydrogenase fragment. Part of these signals are attributed to the reorgan ization of protonated/deprotonated Asp or Glu side chains. On the basis of these data we discuss the role of N2 for proton translocation of complex I.