Regulation of the mechanism of Type-II NADH: Quinone oxidoreductase from S. aureus

Sena, Filipa V.; Sousa, Filipe M.; Oliveira, Ana Sofia F.; Soares, Cláudio M.; Catarino, Teresa; Pereira, Manuela M.

http://hdl.handle.net/10362/65371

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Autores

Sena, Filipa V.

Sousa, Filipe M.

Oliveira, Ana Sofia F.

Soares, Cláudio M.

Catarino, Teresa

Pereira, Manuela M.

Resumo(s)

Type-II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and the only enzymes with NADH:quinone oxidoreductase activity expressed in Staphylococcus aureus (S. aureus), one of the most common causes of clinical infections. NDH-2s are members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, having a flavin adenine dinucleotide, FAD, as prosthetic group and NAD(P)H as substrate. The establishment of a Charge-Transfer Complex (CTC) between the isoalloxazine ring of the reduced flavin and the nicotinamide ring of NAD+ in NDH-2 was described, and in this work we explored its role in the kinetic mechanism using different electron donors and electron acceptors. We observed that CTC slows down the rate of the second half reaction (quinone reduction) and determines the effect of HQNO, an inhibitor. Also, protonation equilibrium simulations clearly indicate that the protonation probability of an important residue for proton transfer to the active site (D302) is influenced by the presence of the CTC. We propose that CTC is critical for the overall mechanism of NDH-2 and possibly relevant to keep a low quinol/quinone ratio and avoid excessive ROS production in vivo.

Descrição

PD/BD/113985/2015 PD/BD/128213/2016 PD/00133/2012 IF/01507/2015 LISBOA-01-0145-FEDER-007660 FVS and FMS are recipients of fellowships by Fundacao para a Ciencia e a Tecnologia (PD/BD/113985/2015, PD/BD/128213/2016, respectively (within the scope of the PhD program Molecular Biosciences PD/00133/2012)). The work was funded by Fundacao para a Ciencia e a Tecnologia (IF/01507/2015 to MMP). This work was also supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI) and by national funds through FCT - Fundacao para a Ciencia e a Tecnologia and by national funds through FCT - Fundacao para a Ciencia e a Tecnologia and by UID/MULTI/04046/2013 centre grant from FCT, Portugal (to BioISI).