Electron Transfer in Subunit NuoI (TYKY) of Escherichia coli NADH:Quinone Oxidoreductase (NDH-1) (original) (raw)

2012, Journal of Biological Chemistry

Background: Subunit NuoI contains two iron-sulfur clusters, N6a and N6b, in the electron transfer pathway of Escherichia coli NDH-1. Results: Contrary to predictions, cluster N6a was EPR-detectable. Conserved residues presumed to be in electron tunneling are not essential. : EPR signals and key residues were assigned. Significance: Missing information on the electron flow was provided. Bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of a peripheral and a membrane domain. The peripheral domain catalyzes the electron transfer from NADH to quinone through a chain of seven iron-sulfur (Fe/S) clusters. Subunit NuoI in the peripheral domain contains two [4Fe-4S] clusters (N6a and N6b) and plays a role in bridging the electron transfer from cluster N5 to the terminal cluster N2. We constructed mutants for eight individual Cys-coordinating Fe/S clusters. With the exception of C63S, all mutants had damaged architecture of NDH-1, suggesting that Cys-coordinating Fe/S clusters help maintain the NDH-1 structure. Studies of three mutants (C63S-coordinating N6a, P110A located near N6a, and P71A in the vicinity of N6b) were carried out using EPR measurement. These three mutations did not affect the EPR signals from [2Fe-2S] clusters and retained electron transfer activities. Signals at g z ‫؍‬ 2.09 disappeared in C63S and P110A but not in P71A. Considering our data together with the available information, g z,x ‫؍‬ 2.09, 1.88 signals are assigned to cluster N6a. It is of interest that, in terms of g z,x values, cluster N6a is similar to cluster N4. In addition, we investigated the residues (Ile-94 and Ile-100) that are predicted to serve as electron wires between N6a and N6b and between N6b and N2, respectively. Replacement of Ile-100 and Ile-94 with Ala/Gly did not affect the electron transfer activity significantly. It is concluded that conserved Ile-100 and Ile-94 are not essential for the electron transfer. The proton-translocating NADH-quinone oxidoreductase (EC 1.6.5.3) (complex I) 3 is the first enzyme in the respiratory chain that couples scalar transfer of electrons from NADH to quinone with vectorial proton translocation across the energytransducing membrane. Mammalian complex I is composed of 45 different subunits and has the most intricate structure of the membrane-bound mitochondrial enzyme complexes (1-3). Dysfunction of complex I has been implicated in numerous human neurodegenerative diseases, including Parkinson disease, Leber hereditary optic neuropathy, and cancer. Complex I is believed to be a major source of reactive oxygen species in mitochondria causing mitochondrial DNA damage and may be one of the causes of aging (4 -6). The bacterial NADH-quinone oxidoreductase enzyme (NDH-1) is simplified version of complex I, which is composed of 13-15 subunits, all of which have homologs in the mitochondrial enzyme . We have shown in a series of publications that the Escherichia coli NDH-1 is a very useful model system to elucidate the structure and function of complex I due to its structural simplicity and ease of gene manipulation (7, 9 -16). Complex I/NDH-1 has a characteristic L-shaped structure with two distinct domains as follows: a hydrophilic peripheral arm projected into the mitochondrial matrix (or bacterial cytoplasm) and a transmembrane hydrophobic arm (17). Recently, the crystal structures of the peripheral and the hydrophobic domains separately, as well as that of the entire complex, have been determined. The revealed structures suggest unprecedented mechanisms for electron transfer and proton translocation (18 -22).