Time-resolved electrochromism associated with the formation of quinone anions in the Rhodobacter sphaeroides R26 reaction center.

The bacterial photosynthetic reaction center contains bacteriochlorophyll (Bchl) and bacteriopheophytin (Bph) cofactors that provide natural probes of electrostatic fields within this protein. We have examined the electrochromic responses of these cofactors, resolved during the lifetimes of the quinone anion states, P+QA-QB and P+QAQB-, and measured as a function of temperature. These measurements provide information on the time-dependent variation in electrostatic field strength on the Bchl and Bph cofactors. Measurements in the near-infrared absorbance bands have revealed the following. First, the QA-QB-->QAQB- electron transfer rate is found to be heterogeneous, consisting of at least two distinct kinetic components. At room temperature, we find a previously unresolved fast kinetic component with a reaction time of 25-40 microseconds, depending upon the preparation, that accounts for approximately 25% of the total reaction yield. The major component was identified with a reaction time of 210-240 microseconds. Below -20 degrees C, QA-QB-->QAQB- electron transfer shows distributed kinetics. The temperature-dependent conversion from biphasic to distributed kinetics suggests that there is a thermal averaging of conformational substates around two reaction center configurations. Interestingly, direct excitation of the Bph with 532 nm light at low temperatures appears to alter the electron transfer kinetics, possibly by inducing a change in the distribution of conformational states. The reaction kinetics were found to be sensitive to the addition of ethylene glycol, which is likely to reflect an osmolarity effect. Second, time-dependent absorption changes of the Bchl and Bph cofactors are found to be kinetically decoupled. The rapid responses of the Bph bands are interpreted to reflect electron transfer, while the slower responses of the Bchl are interpreted to reflect slower relaxation events, possibly including proton uptake. Finally, we find that the electrochromic response and QA-QB-->QAQB- electron transfer to be sensitive to the preparative state of the reaction center, reflecting differences in quinone binding for reaction centers in different states of purification.