Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides

Abstract

The photoreductive trapping of the transient, intermediate acceptor, I −, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I − was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 440, 587–599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301–304). The optical changes of I − showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I − were observed: one was a narrow singlet at g = 2.0035, ΔH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I − on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI −Q −Fe 2+ and the singlet EPR signal is due to DI −Q 2−Fe 2+. ▪ where D is the primary donor (BChl) + 2. The above model was supported by the following observations: 1. (1) During the first illumination, sigmoidal kinetics of the formation of I − was observed. This is a direct consequence of the three-sequential reactions. 2. (2) During the second and subsequent illuminations first-order (exponential) kinetics were observed for the formation of I −. This is due to the dark decay, k 4, to the state DIQ 2−Fe 2+ formed after the first illumination. 3. (3) Removal of the quinone resulted in first-order kinetics. In this case, only the first step, k 1, is operative. 4. (4) The observation of the doublet signal in reaction centers containing menaquinone but not ubiquinone is explained by the longer lifetime of the doublet species I −(Q −Fe 2+) in reaction centers containing menaquinone. The value of τ 2 was determined from kinetic measurements to be 0.01 s for ubiquinone and 4 s for menaquinone ( T = 20°C). The temperature and pH dependence of the dark electron transfer reaction I −(Q −Fe 2+) → I(Q 2−Fe 2+) was studied in detail. The activation energy for this process was found to be 0.42 eV for reaction centers containing ubiquinone and 0.67 eV for reaction centers with menaquinone. The activation energy and the doublet splitting were used to calculate the rate of electron transfer from I − to MQ −Fe 2+ using Hopfield's theory for thermally activated electron tunneling. The calculated rate agrees well with the experimentally determined rate which provides support for electron tunneling as the mechanism for electron transfer in this reaction. Using the EPR doublet splitting and the activation energy for electron transfer, the tunneling matrix element was calculated to be 10 −3 eV. From this value the distance between I − and MQ − was estimated to be 7.5–10 Å.