Conformational dynamics of proton-translocation in the respiratory complex I
Final Report Abstract
The proton-pumping NADH:ubiquinone oxidoreductase, also known as respiratory complex I, is a major enzyme in energy metabolism in most cells. In humans, its dysfunction is connected to many neurodegenerative diseases such as Parkinsons disease. Complex I is the first enzyme of respiratory chains. It transfers electrons from NADH to the membrane-bound ubiquinone. The electron transfer reaction is coupled with a translocation of protons across the membrane. In doing so, complex I contributes to the proton motive force required for energy consuming processes, like ATP synthesis. While the structures of the other enzyme complexes of the respiratory chains have been determined at molecular resolution, little is known about the structure of complex I. Electron microscopy revealed the 'L'-shape of the complex consisting of two 200 A long arms arranged perpendicular to each other. A peripheral arm protudes into the aqueous phase while a membrane arm is buried in the lipid bilayer. The bacterial complex I consisting in general of 14 different subunits, is considered to be a structural minimal form of complex I. Seven subunits are peripheral proteins including the subunits that bear all known redox groups, namely one flavin mononucleotide (FMN) and up to nine iron-sulfur (Fe/S) clusters. These subunits build the peripheral arm. Seven subunits are highly hydrophobic proteins predicted to fold into 61 a-helices across the membrane. They build the membrane arm. So far no redox groups have been detected in these subunits, which have to be involved in proton translocation. Recently, the structure of the peripheral arm of complex I from Thermus thermophilus was resolved revealing the positions of the cofactors. Our group had proposed a novel mechanism wherein the electron transfer reaction induces conformational changes which subsequently lead to the translocation of protons. To substantiate this proposal, we first determined the type of ion translocated by the E. coli complex I, which is essential for the enzyme mechanism. It was proposed that the complex works not as a proton but a sodium pump. However, we clearly demonstrated that the bacterial complex I like its mitochondrial homologue works as a primary proton pump. There is some indication that the redox reaction might be coupled to a secondary sodium/proton antiport. We showed that the proton translocation is coupled to the electron transfer between the most distal Fe/S cluster N2 and the substrate ubiquinone. This redox reaction induces the protonation/deprotonation of amino acid residues in its vicinity. Mutation of these amino acid residues to residues, which are not able to perform protonation/deprotonation steps led to an inhibition of the electron transfer reaction indicating the strong coupling between the redox reaction and the proton transfer reaction. The step of the redoxreaction that induced the protonation of individual amino acid residues also led to conformational changes of the complex as detected by FT-IR spectroscopy. The amplitude of the signal is pHdependent as expected for an energy coupling reaction. The enzymes conformational changes led to a change of the environment of bound lipid(s) indicating a participation of lipids to this process by a yet unknown mechanism. Electron microscopy and CD spectroscopy revealed that an addition of NADH but not NADPH led to conformational changes of complex I, although all Fe/S clusters of the complex are rapidly reduced by NADH and NADPH. From this we concluded that the binding of NADH but not NADPH induces conformational changes that open the quinone binding site. The effect of NADH on the conformation of the complex was corroborated by EPR spectra of complex I covalently labeled with an MTSL spin label at particular positions recorded in the presence and the absence of NADH. Addition of NADH but not NADPH led to a change of the environment of the spin label depending on the the position of the label.
Publications
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Flemming, D., Hellwig, P., Lepper, S., Kloer, D,P,, and Friedrich, T. (2006) Catalytic Importance of Acidic Amino Acids on Subunit NuoB of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I). J. Biol. Chem. 281, 24781-24789.
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Friedrich, T., Stolpe, S., Schneider, D., Barquera, B., and Hellwig, P. (2005) Ion translocation by the Escherichia coli NADH:ubiquinone oxidoreductase (complex I). Biochem. Soc. Trans. 33,836-839.
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Hellwig, P., Stolpe, S., and Friedrich, T. (2004) FTIR spectroscopic study on the conformational reorganization in E, coli complex I due to redox-driven proton translocation. Biopolymers 74, 69-72.
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Hielscher, R., Wenz, T., Stolpe, S., Hunte, C., Friedrich, T., and Hellwig, P. (2006) Monitoring redox dependent contribution of lipids in FTIR difference spectra of complex I from E, coli. Biopolymers 82, 291-294.
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Pohl, T., Bauer, T., Dörner, K., Stolpe, S., Sell, P., Zocher, G., and Friedrich, T. (2007) Iron-sulfur cluster N7 of the NADH:ubiquinone oxidoreductase (complex I) is essential for stability but not involved in electron transfer. Biochemistry 46, 6588-6596.
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Stolpe, S. and Friedrich, T. (2004) The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton-pump but may be capable of secondary sodium antiport. J. Biol. Chem. 279, 18377-18383.
