Abstract
The bacterial H + -translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1-3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue ( K Glu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue ( K Glu-72 in TM3) moderately reduced the activities. To clarify the contribution of Nuo K to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted K Glu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of K Glu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK ( K Arg-25, K Arg-26, and K Asn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.