Photosynthesis Research 70: 175–184, 2001. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 175 Regular paper Photoinhibition of carotenoidless reaction centers from Rhodobacter sphaeroides by visible light. Effects on protein structure and electron transport J´ ulia Tandori 1 , ´ Eva Hideg 2 , L´ aszl´ o Nagy 1 , P´ eter Mar´ oti 1 & Imre Vass 2,∗ 1 Department of Biophysics, University of Szeged, Hungary; 2 Institute of Plant Biology, Biological Research Center P.O. Box 521, H-6701 Szeged, Hungary; ∗ Author for correspondence (e-mail: imre@nucleus.szbk.u-szeged.hu; fax: +36-62-433-434) Received 10 January 2001; accepted in revised form 30 May 2001 Key words: bacterial reaction center, carotenoids, photoinhibition, protein damage, singlet oxygen Abstract Inhibition of electron transport and damage to the protein subunits by visible light has been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides. Illumination by 1100 μEm -2 s -1 light induced only a slight effect in wild type, carotenoid containing 2.4.1. reaction centers. In contrast, illumination of reaction centers isolated from the carotenoidless R26 strain resulted in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm arising from the P + Q B - → PQ B recombination. In addition to this effect, the L, M and H protein subunits of the R26 reaction center were damaged as shown by their loss on Coomassie stained gels, which was however not accompanied by specific degradation products. Both the loss of photochemical activity and of protein subunits were suppressed in the absence of oxygen. By applying EPR spin trapping with 2,2,6,6-tetramethylpiperidine we could detect light-induced generation of singlet oxygen in the R26, but not in the 2.4.1. reaction centers. Moreover, artificial generation of singlet oxygen, also led to the loss of the L, M and H subunits. Our results provide evidence for the common hypothesis that strong illumination by visible light damages the carotenoidless reaction center via formation of singlet oxygen. This mechanism most likely proceeds through the interaction of the triplet state of reaction center chlorophyll with the ground state triplet oxygen in a similar way as occurs in Photosystem II. Abbreviations: Bchl – bacteriochlorophyll; P – Bchl dimer; DMPO – 5,5-dimethyl-1-pyrroline N-oxide; Q A – primary quinone electron acceptor; Q B – secondary quinone electron acceptor; RC – reaction center; TEMPO – 2,2,6,6-tetramethyl-piperidine-1-oxyl; ROS – reactive oxygen species Introduction Light is a well-known damaging factor of the pho- tosynthetic apparatus in oxygen evolving organisms in which the Photosystem II (PS II) complex is the primary target site (for reviews see Andersson and Styring 1991; Andersson et al. 1992; Aro et al. 1993). The main mechanism of photoinhibitory damage is the production of highly reactive singlet oxygen. This pro- cess is initiated by a light-induced block of electron transfer between the primary Q A and secondary Q B quinone electron acceptors, which leads to the double reduction of Q A . This in turn promotes the forma- tion of triplet reaction center (RC) chlorophyll, which produces singlet oxygen via interaction with ground state triplet oxygen (Durrant et al. 1990; Vass et al. 1992; Hideg et al. 1994b). Photoinhibition of PS II electron transport is followed by the degradation of the D1 and, to a lesser extent, of the D2 reaction center subunits (Kyle et al. 1984; Ohad et al. 1984; Prasil et al. 1992), which is initiated by oxidative damage by singlet oxygen (Aro et al. 1993; Hideg et