Alternative Sites for Proton Entry from the Cytoplasm to the Quinone Binding Site in Escherichia coli Succinate Dehydrogenase

Abstract

Escherichia coli succinate dehydrogenase (Sdh) belongs to the highly conserved complex II family of enzymes that reduce ubiquinone. These enzymes do not generate a protonmotive force during catalysis and are electroneutral. Because of its electroneutrality, the quinone reduction reaction must consume cytoplasmic protons which are released stoichiometrically during succinate oxidation. The X-ray crystal structure of E. coli Sdh shows that residues SdhB (G227), SdhC (D95), and SdhC (E101) are located at or near the entrance of a water channel that has been proposed to function as a proton wire connecting the cytoplasm to the quinone binding site. However, the pig and chicken Sdh enzymes show an alternative entrance to the water channel via the conserved SdhD (Q78) residue. In this study, site-directed mutants of these four residues were created and characterized by in vivo growth assays, in vitro activity assays, and electron paramagnetic resonance spectroscopy. We show that the observed water channel in the E. coli Sdh structure is the functional proton wire in vivo, while in vitro results indicate an alternative entrance for protons. In silico examination of the E. coli Sdh reveals a possible H-bonding network leading from the cytoplasm to the quinone binding site that involves SdhD (D15). On the basis of these results we propose an alternative proton pathway in E. coli Sdh that might be functional only in vitro.

Structure of Escherichia coli Succinate:Quinone Oxidoreductase with an Occupied and Empty Quinone-binding Site

“…The orientation of carboxin in the Q-site differs with computational predictions (16) and with that seen in avian SQR (2FBW). The position of conserved water molecules around the Q-site suggests a new water-mediated proton uptake pathway consistent with recent mutational and biophysical studies (19).…”

“…5 The conservation of the waters shown in Fig. 5C and the similar location of possible water channels in eukaryotic SQRs (19) are consistent with some involvement of the water chain in protonation/deprotonation reactions of quinones. In E. coli the SQR trimer may also provide a water sink to provide bulk solvent water for these reactions.…”

“…These water molecules have an average B-factor of 46.7 Å 2 , comparable with the average B-factor of the protein, showing that the water molecules are well ordered. A recent mutagenesis study also suggested a possible alternative entrance for protons involving Asp-D15 (19). In Fig.…”

“…5C the waters have been colored to reflect their degree of conservation within the three NCS copies of the monomer; dark green for those waters conserved in all three copies (B2103, D2008, B2120, and D2001), chartreuse for the one present in two copies (B2123), and lime green for the water present in one copy (D2003). Significantly, waters D2003 and D2008 hydrogen-bond to Gln-D78, which lies on the entrance to possible water channels identified in the porcine and avian SQR structures (19).…”

A conserved lysine residue controls iron–sulfur cluster redox chemistry in Escherichia coli fumarate reductase

“…4B). The WT Sdh enzyme exhibits a broad signal that comprises two overlapping peaks located at g = 3.66 and g = 3.55 [6,39,40]. In the SdhB-R205S variant, the two components observed in the wild-type enzyme collapse into a single archetypal highly anisotropic low spin (HALS) heme spectrum with a single ramp-type feature corresponding to g z [41,42].…”