Sena, Filipa V.Sousa, Filipe M.Pereira, Ana R.Catarino, TeresaCabrita, Eurico J.Pinho, Mariana G.Pinto, Francisco R.Pereira, Manuela M.2024-10-292024-10-292024-082165-0497PURE: 99131336PURE UUID: 062ed43f-df89-482b-ab51-de6c7bf9a88dScopus: 85201029856WOS: 001268104800001PubMed: 39012110PubMedCentral: PMC11302666http://hdl.handle.net/10362/174200Funding Information: F.V.S. and F.M.S. were recipients of fellowships by Fundação para a Ciência e a Tecnologia within the scope of the PhD program Molecular Biosciences PD/00133/2012. Publisher Copyright: © 2024 Sena et al.Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to differentniches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavinis inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently.The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.242998004engalternative NADH oxidasecharge-transfer complexmembrane proteinsmonotopic proteinsNAD(P)Hquinonesrespiratory chainPhysiologyEcologyGeneral Immunology and MicrobiologyGeneticsMicrobiology (medical)Cell BiologyInfectious DiseasesSDG 3 - Good Health and Well-beingjournal article10.1128/spectrum.04152-23https://www.scopus.com/pages/publications/85201029856