Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: Implications for the mechanism of electron and proton transfer in proteins

Citation
Ms. Graige et al., Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: Implications for the mechanism of electron and proton transfer in proteins, BIOCHEM, 38(35), 1999, pp. 11465-11473
Citations number
57
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
38
Issue
35
Year of publication
1999
Pages
11465 - 11473
Database
ISI
SICI code
0006-2960(19990831)38:35<11465:OOTPSI>2.0.ZU;2-B
Abstract
A proton-activated electron transfer (PAET) mechanism, involving a protonat ed semiquinone intermediate state, had been proposed for the electron-trans fer reaction k(AB)((2)) [Q(A) (-.)Q(B)(-.) + H+ reversible arrow Q(A)(-.)(Q (B)H)(.) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sph aeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamu ra, M. Y. (1996) J. Am. Chem, Soc. 118, 9005-9016], Confirmation of this me chanism by observing the protonated semiquinone (Q(B)H)(.) had not been pos sible, presumably because of its low pK(a). By replacing the native Q(10) i n the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were a ble to observe the (Q(B)H)(.) state. The pH dependence of the semiquinone o ptical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rat e for the k(AB)((2)) reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-.) to the protonated semiquinone (i.e., k (AB)((2)) = 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 pro ton-transfer rate k(H) >> 10(4) s(-1). Applying the two step mechanism to R Cs containing native Q(10) and taking into account the change in redox pote ntial, we find reasonable values for the fraction of (QBH). congruent to 0. 1% (consistent with a pK(a)(Q(10)) of similar to 4.5) and k(ET)(Q(10)) cong ruent to 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.