The two alternative NADH:quinone oxidoreductases from : two players with different molecular and cellular roles.

is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of , focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. 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 (Δ and Δ). 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 flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP and FADH, 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.IMPORTANCE is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow to live and survive. Despite its prominence as a pathogen, the energy metabolism of remains underexplored, with its respiratory enzymes often escaping thorough investigation. bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.