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The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c1, which form two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the pathways of bifurcated electron transfers emanating from QH2 oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn2+ binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc1. In this work, using the genetically tractable bacterial cyt bc1, we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Qo site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH2 oxidation at the Qo site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH2 oxidation at the Qo site of cyt bc1.

The photosynthetic reaction center (RC) of Rb. sphaeroides catalyzes light-induced electron transfer events which are connected to the conformational dynamics of the protein. The light-induced charge separation between the primary donor (P) and the quinone acceptor (QA) is stabilized by solvent/protein conformational rearrangements. After a laser pulse P+QA recombination, which occurs with a lifetime t~100 ms in room temperature solutions, is accelerated (t~20 ms) at cryogenic temperatures [1] and in dehydrated glassy matrices at room temperature [2]. After prolonged photoexcitation, a slow phase of recombination (t~250 s) is observed, attributed to additional conformational changes [3]. Differential FTIR bands of water associated with the QA/QA transition have been observed upon continuous illumination, leading to propose that weakly bound water molecules plays a role in P+QA stabilization [4]. By controlling the hydration level of RC-detergent films, through equilibration at given relative humidities (r), a strong inhibition of the P+QA conformational stabilization has been observed at low hydration [5]. We compared FTIR light-minus-dark (P+QA/ PQA) differential spectra in hydrated (r=76%) and dehydrated (r=11%) RC films over the 4000- 1000 cm-1. The spectra differ significantly in the 3750-3550 cm-1 range, the band attributed to weakly hydrogen bonded water molecules [5] being strongly reduced in the dried film. Dehydration also affects the 1800-1200 cm-1 range, which includes contributions from P, the quinones and the peptide. Optical absorption measurements performed under the same photoexcitation regime reveal a slow (t~5 s) kinetic component of P+QA recombination which disappears in the dehydrated sample, indicating at low r a destabilization of the charge separated state. As a whole the data suggest a correlation between the hydration shell dynamics and the conformational RC dynamics which stabilize the charge separated state.

The interplay between protein dynamics and electron transfer (ET) has been extensively investigated in the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides by hampering RC internal motions at low temperatures [1]. Alternatively, the RC dynamics can be inhibited at room temperature by incorporating the RC into dehydrated trehalose matrices [2]. In the glasses the recombination kinetics of the charge separated state P+QA are accelerated and distributed in rate as compared to solution, mimicking at room temperature the effects observed at 10 K in water-glycerol. This is taken to indicate inhibition of the RC relaxation from the dark- to the light-adapted conformation, as well as of the RC thermal fluctuations [1,2]. We proposed that the inhibition is mediated by residual water molecules of the RC hydration shell which bridge protein surface groups with trehalose molecules of the matrix by forming a network of multiple hydrogen bonds [3]. Consistently, similar effects have been observed also in RC films dehydrated in the absence of sugar [4]. However, striking differences are found between RC-trehalose glasses and RC-films: (a) The thermal stability of the RC is tremendously enhanced in the trehalose matrix. (b) In RC-trehalose matrices, the P+QA recombination after a few seconds of continuous photoexcitation is only partially decelerated as compared to the one recorded after a laser flash, whereas in dried RC films a comparable period of continuous illumination leads to a total recovery of the kinetics observed in the hydrated system. These data indicate that in films the protein dynamics can be easily regained, in contrast to trehalose glasses, which reveal much stronger structural constraints. We are extending these studies to a series of RC mutants characterized by a widely altered P+/P midpoint potential relative to wild-type [5]. In these mutants, the acceleration of P+QA recombination induced by cooling to 10 K in the dark decreased with increasing midpoint potential [6]. Interestingly, incorporation into dehydrated trehalose matrices causes instead acceleration of the kinetics by the same factor for all P+/P midpoint potential values.