Trapping Conformational Intermediate States in the Reaction Center Protein from Photosynthetic Bacteria

Abstract

In protein, conformational changes are often crucial for function but not easy to observe. Two functionally relevant conformational intermediate states of photosynthetic reaction center protein (RCs) are trapped and characterized at low temperature. RCs frozen in the dark do not allow electron transfer from the reduced primary quinone, Q A -, to the secondary quinone, Q B . In contrast, RCs frozen under illumination in the product (P + Q A Q B -) state, with the oxidized electron donor, P + , and reduced Q B -, return to the ground state at cryogenic temperature in a conformation that allows a high yield of Q B reduction. Thus, RCs frozen under illumination are found to be trapped above the ground state in a conformation that allows product formation. When the temperature is raised above 120 K, the protein relaxes to an inactive conformation which is different from the RCs frozen in the dark. The activation energy for this change is 87 ( 8 meV, and the active and inactive states differ in energy by only 16 ( 3 meV. Thus, there are several conformational substates along the reaction coordinate with different transition temperatures. The ground state spectra of the RCs in active and inactive conformations report differences in the intraprotein electrostatic field, demonstrating that the dipole or charge distribution has changed. In addition, the electrochromic shift associated with the Q A -to Q B electron transfer at low temperature was characterized. The electron-transfer rate from Q B -to P + was measured at cryogenic temperature and is similar to the rate at room temperature, as expected for an exothermic, electron tunneling reaction in RCs.Conformational flexibility is important for the function of proteins (1, 2). At room temperature, proteins fluctuate among many conformational states. At low temperature, proteins will become trapped in harmonic motions near the conformation it was frozen into (3-9). Intraprotein reactions are inhibited if this conformation cannot access the transition state. When the temperature is raised, relaxation between conformational substates becomes possible. Elastic incoherent neutron scattering and X-ray crystallography measurements of bacteriorhodopsin (10) and myoglobin (4) show that different parts of the protein relax at different temperatures (11).Bacterial photosynthetic reaction center protein (RCs) 1 has a number of physiological electron tunneling reactions that occur even at cryogenic temperatures. This system can therefore be used to characterize conformational substates of physiologically important reactions. Previous studies of RCs have characterized the protein at cryogenic temperatures. RCs can be trapped in a distribution of substates which exhibit a wider distribution of reaction rates (12,13). Other studies have characterized unrelaxed product states (14). In addition, reactions that normally do not occur at low temperatures have been observed when the protein is frozen into appropriate conformational states (12).The bacterial photosynthetic reaction c...

ENDOR Spectroscopy Reveals Light Induced Movement of the H-Bond from Ser-L223 upon Forming the Semiquinone (QB-•) in Reaction Centers fromRhodobacter sphaeroides

“…Similar long lived states have been observed by other authors for native RCs frozen under different illumination conditions (15,18,38,41,42). In principal the longer lifetime of the long lived state frozen in the light may be due to a reduced electron transfer rate for the back electron transfer reaction.…”

“…In early studies, large differences in transfer rates were observed between RCs frozen in the light or in the dark (14,15,18). For RCs frozen in the dark and illuminated at cryogenic temperatures, the reaction k AB…”

“…For RCs frozen in the dark and illuminated at cryogenic temperatures, the reaction k AB (1) , became slower as the temperature decreased and did not appreciably occur below 150K. However, if RCs were illuminated during the freezing process in the D +• Q B −• state and the charges were allowed to recombine without thawing, photochemical electron transfer at cryogenic temperatures was observed (15,18), showing that a change in a structure has occurred between the neutral state and the charge separated state. The x-ray crystal structure of RCs frozen in the light (Q B The large change in the position of the quinone head group suggested that the movement of Q B from the distal to the proximal position is a possible mechanism for conformational gating of the electron transfer to Q B (8,9).…”

Trapped Conformational States of Semiquinone (D+•QB-•) Formed by B-Branch Electron Transfer at Low Temperature in Rhodobacter sphaeroides Reaction Centers

“…In this study, electron transfer rates were measured for RCs frozen in different states at cryogenic temperature (5,43,63,64). s with a fraction that did not recover, attributed to samples that may have lost the electron on the acceptor side.…”

“…Subsequently several studies to investigate these conformers (i.e. substates) were performed and the properties of charge separation investigated (43,73). These states have been proposed in a general sense to differ in the detailed interactions between Q B and the protein.…”