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.

Membrane protein complexes can support both the generation and utilization of a transmembrane electrochemical proton potential (Deltap), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. Regarding the first mechanism, this has been unequivocally demonstrated to be operational for Deltap-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing SQR (succinate:menaquinone reductase) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane Deltap to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing QFR (quinol:fumarate reductase) of the epsilon-proteobacterium Wolinella succinogenes, evidence has been obtained indicating that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway ('E-pathway'). This is necessary because, although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Deltap. This compensatory E-pathway appears to be required by all dihaem-containing QFR enzymes and the conservation of the essential acidic residue on transmembrane helix V (Glu-C180 in W. succinogenes QFR) is a useful key for the sequence-based discrimination of these QFR enzymes from the dihaem-containing SQR enzymes.

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To gain new insights into the function of photosystem II (PSII) herbicides DCMU (a urea herbicide) and bromoxynil (a phenolic herbicide), we have studied their effects in a better understood system, the bacterial photosynthetic reaction center of the terbutryn-resistant mutant T4 of Blastochloris (Bl.) viridis. This mutant is uniquely sensitive to these herbicides. We have used redox potentiometry and time-resolved absorption spectroscopy in the nanosecond and microsecond time scale. At room temperature the P(+)(*)Q(A)(-)(*) charge recombination in the presence of bromoxynil was faster than in the presence of DCMU. Two phases of P(+)(*)Q(A)(-)(*) recombination were observed. In accordance with the literature, the two phases were attributed to two different populations of reaction centers. Although the herbicides did induce small differences in the activation barriers of the charge recombination reactions, these did not explain the large herbicide-induced differences in the kinetics at ambient temperature. Instead, these were attributed to a change in the relative amplitude of the phases, with the fast:slow ratio being approximately 3:1 with bromoxynil and approximately 1:2 with DCMU at 300 K. Redox titrations of Q(A) were performed with and without herbicides at pH 6.5. The E(m) was shifted by approximately -75 mV by bromoxynil and by approximately +55 mV by DCMU. As the titrations were done over a time range that is assumed to be much longer than that for the transition between the two different populations, the potentials measured are considered to be a weighted average of two potentials for Q(A). The influence of the herbicides can thus be considered to be on the equilibrium of the two reaction center forms. This may also be the case in photosystem II.

Photosynthetic reaction centers of Blastochloris viridis require two quanta of light to catalyse a two-step reduction of their secondary ubiquinone Q B to ubiquinol. We employed capacitive potentiometry to follow the voltage changes that were caused by the accompanying transmembrane proton displacements. At pH 7.5 and 20°C, the Q B -related voltage generation after the first flash was contributed by a fast, temperature-independent component with a time constant of $30 ls and a slower component of $200 ls with activation energy (E a ) of 50 kJ/mol. The kinetics after the second flash featured temperature-independent components of 5 ls and 200 ls followed by a component of 600 ls with E a $ 60 kJ/mol.