Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at A resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called "E-pathway hypothesis". According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme-propionate involvement in redox-coupled proton transfer by a combination of specific (13)C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme-precursor 5-aminolevulinate and by providing [1-(13)C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfer.
The water is slow to exchange, showing the dynamic behavior of bulk water 25 °C colder [ 147 ]. Low-density water (such as ES ) is promoted [ 148 , 276 ] surrounding this dense hydration and polyelectrolyte double layer (as described in the ' Polysaccharide hydration ' section). Non-polar groups promote clathrate structures [ 153 ] (such as ES ) surrounded by denser water. It is no surprise, therefore that the degree of hydrophobic hydration is correlated with the hydration of the polar groups further away. As clathrate-type structures break down at higher temperatures, hydrophobic hydration shows greater temperature dependence than hydrophilic hydration [ 2761 ]. Clathrate shells contain loosely held water with greater rotational freedom than in the bulk [ 139 ]. However under favorable conditions, clathrate hydrophobic hydration may exert pressure on non-polar C-H bonds pushing them in, so contracting their bond length and increasing their vibrational frequency. This blue-shifting (that is, the vibration frequency increases and intensity reduces) 'push-ball' hydration [ 149 ] should not necessarily be thought of as 'typical' hydrogen bonding even if the CH···OH 2 distances are suitably close (see [ 1293 ]). They can be considered as part of a continuum of hydrogen bonding behavior, however, where sometimes the OH 2 behaves as a much more weakly interacting base than usual and the C-H behaves with reversed dipolar behavior compared with the more usual O-H hydrogen-bonding partners [ 625 ].