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