Hydrogen-Bonded Networks Along and Bifurcation of the E-Pathway in Quinol:Fumarate Reductase

Abstract

The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme b(D) ring C (b(D)-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the b(D)-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the b(D)-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme b(D) ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.

The di-heme family of respiratory complex II enzymes

“…The results obtained indicated that transiently established hydrogen bonding paths involving internal water molecules are responsible for proton transfer from the heme b D ring-C-propionate to Glu C180 (Fig. 7A,B) and that high proton conductance associated with short hydrogen bonding paths could be identified only for the reduced but not the oxidized state of the enzyme [44]. These differences were also associated with a conformational change in the orientation of the Glu C180 side chain in agreement with the predictions from multiconformation continuum electrostatics calculations [45], results from FTIR difference spectroscopy [61] and with alternate conformers in the 1.78 Å resolution crystal structure (PDB ID: 2BS2 [33]).…”

“…It has long been known [42] that the two heme groups have different oxidation-reduction midpoint potentials and an assignment for B. subtilis SQR based on the effects of site-directed mutagenesis has been made [43]. Although in retrospect this assignment is probably correct, the interpretation of such mutagenesis effects can be misleading, as demonstrated recently [44] by the stronger effect of the replacement of a W. succinogenes QFR heme b L ligand on the midpoint potential of heme b H rather than on that of heme b L . A mutagenesis-independent assignment of the "high-potential" heme to b P and the "low-potential" heme to b D was achieved on the basis of structure-based electrostatic calculations [45].…”

“…However, functionality of the E-pathway requires the enzyme to be reduced [60]. Lacking an experimentally determined structure of the reduced enzyme, Herzog et al [44] performed molecular dynamics simulations of various redox states of the enzyme. The results obtained indicated that transiently established hydrogen bonding paths involving internal water molecules are responsible for proton transfer from the heme b D ring-C-propionate to Glu C180 (Fig.…”

“…Taken [44]) Superposition of an MD snapshot without hydrogen bonding paths (helix and carbon atoms coloured in white) and one with formed hydrogen bonding paths (helix and carbon atoms coloured in yellow). Side chains of His C44 (labeled "H44"), a bridging water molecule as well as the carboxyl group of the heme b D propionate are highlighted by using thicker sticks.…”

“…In compensation, coupled to electron transfer via the two heme groups, protons are transferred from the periplasm via the ring C propionate of the distal heme b D and the residue Glu C180 (indicated by the blue circles) to the cytoplasm (top), where they replace those protons which are bound during fumarate reduction. One of these protons is taken up from the periplasm, the other transferred directly from the menaquinol oxidation site [44]. In the oxidized state of the enzyme, the "E-pathway" is blocked.…”