The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.

离子泵 NQR 复合物是许多病原菌生理学中必不可少的呼吸酶。这种酶通过包括核黄素（维生素 B2）在内的几种辅助因子将电子从 NADH 转移到泛醌。NQR 是唯一一种能够使用核黄素作为辅因子的酶。此外，核黄素分子被发现是一种稳定的中性半醌自由基。否则高反应性的未配对电子通过稳定一种未知的机制。晶体学数据表明核黄素可能存在于 NQR 亚基 B 和 E 界面的表面位置。然而，该位置存在很大问题，因为该位置不具有预期的理化特性。在这项工作中，我们将核黄素结合位点定位在亚基 B 的两亲口袋中，之前提出是钠的进入位点。在这里，我们显示该站点包含绝对保守的残基，包括 N200、N203 和 D346。这些残基的突变会降低酶活性并特异性阻断 NQR 结合核黄素的能力。对接分析和分子动力学模拟表明，这些残基直接参与核黄素结合，建立稳定该位点辅助因子的氢键。