Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: implications for the mechanism of electron and proton transfer in proteins.
A proton-activated electron transfer (PAET) mechanism, involving a protonated semiquinone intermediate state, had been proposed for the electron-transfer reaction k(2)AB [Q(A)(-)(*)Q(B)(-)(*) + H(+) <--> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sphaeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016]. Confirmation of this mechanism by observing the protonated semiquinone (Q(B)H)(*) had not been possible, presumably because of its low pK(a). By replacing the native Q(10) in the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were able to observe the (Q(B)H)(*) state. The pH dependence of the semiquinone optical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rate for the reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-)(*) to the protonated semiquinone (i.e., k(2)AB = k(ET)(RQ) = 2 x 10(4) s(-)(1)). The rate decreased at pH > pK(a) as predicted by the PAET mechanism in which fast reversible proton transfer precedes rate-limiting electron transfer. Consequently, near pH 7, the proton-transfer rate k(H) >> 10(4) s(-)(1). Applying the two step mechanism to RCs containing native Q(10) and taking into account the change in redox potential, we find reasonable values for the fraction of (Q(B)H)(*) congruent with 0.1% (consistent with a pK(a)(Q(10)) of approximately 4.5) and k(ET)(Q(10)) congruent with 10(6) s(-)(1). These results confirm the PAET mechanism in RCs with RQ and give strong support that this mechanism is active in RCs with Q(10) as well.
Graige
M S
MS
Department of Physics, University of California, San Diego, La Jolla 92093-0319 USA.
Paddock
M L
ML
Feher
G
G
Okamura
M Y
MY
D016227:Benzoquinones / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009285:Naphthoquinones / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010782:Photolysis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D018892:Proton-Motive Force; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D016227:Benzoquinones; D009285:Naphthoquinones; D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D014451:Ubiquinone; C025232:semiquinone radicals; C003304:rhodoquinone