Identification of the Proton Pathway in Bacterial Reaction Centers:  Cooperation between Asp-M17 and Asp-L210 Facilitates Proton Transfer to the Secondary Quinone (Q_B)

Abstract

The reaction center (RC) from Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, Q(B) (the secondary quinone electron acceptor), to form quinol, Q(B)H2. Asp-L210 and Asp-M17 have been proposed to be components of the pathway for proton transfer [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. To test the importance of these residues for efficient proton transfer, the rates of the proton-coupled electron-transfer reaction k(AB)(2) (Q(A-*)Q(B-*) + H+ <==>Q(A-*)Q(B)H* --> Q(A)Q(B)H-) and its associated proton uptake were measured in native and mutant RCs, lacking one or both Asp residues. In the double mutant RCs, the k(AB)(2) reaction and its associated proton uptake were approximately 300-fold slower than in native RCs (pH 8). In contrast, single mutant RCs displayed reaction rates that were < or =3-fold slower than native (pH 8). In addition, the rate-limiting step of k(AB)(2) was changed from electron transfer (native and single mutants) to proton transfer (double mutant) as shown from the lack of a dependence of the observed rate on the driving force for electron transfer in the double mutant RCs compared to the native or single mutants. This implies that the rate of the proton-transfer step was reduced (> or =10(3)-fold) upon replacement of both Asp-L210 and Asp-M17 with Asn. Similar, but less drastic, differences were observed for k(AB)(1), which at pH > or =8 is coupled to the protonation of Glu-L212 [(Q(A-*)Q(B))-Glu- + H+ --> (Q(A)Q(B-*)-GluH]. These results show that the pathway for proton transfer from solution to reduced Q(B) involves both Asp-L210 and Asp-M17, which provide parallel branches to the proton-transfer pathway and through their electrostatic interaction have a cooperative effect on the proton-transfer rate. A possible mechanism for the cooperativity is discussed.

Energetics of Proton Transfer Pathways in Reaction Centers from Rhodobacter sphaeroides

“…Contrary to the revertant mutants, the inhibited mutants and the AA mutants showed a pK a for Glu-H173 that is 1.1 and 3.2 units lower than in the wild type bRC, respectively (Table II). In the wild type bRC, the calculated pK a of 5.4 for Asp-M17 is significantly larger than that of 2.7 for Glu-H173, which favors an involvement of Asp-M17 rather than Glu-H173 in the PT pathway (44,46). Notably, all mutant bRC considered here showed a pK a decrease for Asp-M17 by 1.4 -2.4 units with respect to wild type bRC (Table II).…”

Proton transfer in bioenergetic proteins

“…In contrast, the PT mutants (L213DN single mutant and L210DN/M17DN double mutant together with native RC poisoned by transient divalent ions) show much larger (up to 4 orders of magnitude) decrease relative to that of the native value (Fig. 3b) in nice agreement with earlier measurements [20,26].…”

“…It is worth to carry out similar isotope measurements with the 2nd ET of various proton transfer RC variants. These RCs impede the normal fast function of the bucket brigade mechanism of PT at well defined locations: native RC treated with divalent metal ions at the proton entry point [24,25], L210DN/M17DN double mutation between L210D and M17D [26,27] and L213DN single mutation at L213 close (b5 Å) to Q B [20,28]. The proton delivery with significantly increased free energy of activation will be the bottle neck of the observed 2nd ET (Fig.…”

“…The 420 nm band of the spectra at low pH resembles the protonated spectrum of semiquinone in solution. 26 The spectra are normalized to the absorption at 450 nm. study than that reported previously.…”

My Road to Biophysics: Picking Flowers on the Way to Photosynthesis

“…We used the semiconductor InSb and measured the nuclear polarizations at 4.2 K of In 115 , Sb 121 , and Sb 123 in the presence of an electric field. The nuclear relaxation times were several hours, and we ran the experiments uninterrupted for ∼24 h. Nuclear polarization enhancements of up to 100 were obtained in agreement with theoretical predictions.…”