Structural Characterization of HP1264 Reveals a Novel Fold for the Flavin Mononucleotide Binding Protein

Complex I (NADH-quinone oxidoreductase) is an enzyme that catalyzes the initial electron transfer from nicotinamide adenine dinucleotide (NADH) to flavin mononucleotide (FMN) bound at the tip of the hydrophilic domain of complex I. The electron flow into complex I is coupled to the generation of a proton gradient across the membrane that is essential for the synthesis of ATP. However, <i>Helicobacter pylori</i> has an unusual complex I that lacks typical NQO1 and NQO2 subunits, both of which are generally included in the NADH dehydrogenase domain of complex I. Here, we determined the solution structure of HP1264, one of the unusual subunits of complex I from <i>H. pylori</i>, which is located in place of NQO2, by three-dimensional nuclear magnetic resonance (NMR) spectroscopy and revealed that HP1264 can bind to FMN through UV–visible, fluorescence, and NMR titration experiments. This result suggests that FMN-bound HP1264 could be involved in the initial electron transfer step of complex I. In addition, HP1264 is structurally most similar to <i>Escherichia coli</i> TusA, which belongs to the SirA-like superfamily having an IF3-like fold in the SCOP database, implying that HP1264 adopts a novel fold for FMN binding. On the basis of the NMR titration data, we propose the candidate residues Ile32, Met34, Leu58, Trp68, and Val71 of HP1264 for the interaction with FMN. Notably, these residues are not conserved in the FMN binding site of any other flavoproteins with known structure. This study of the relationship between the structure and FMN binding property of HP1264 will contribute to improving our understanding of flavoprotein structure and the electron transfer mechanism of complex I