TIME-RESOLVED ELECTROCHROMISM ASSOCIATED WITH THE FORMATION OF QUINONE ANIONS IN THE RHODOBACTER-SPHAEROIDES R26 REACTION-CENTER

The bacterial photosynthetic reaction center contains bacteriochloroph yll (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 l ifetimes of the quinone anion states, P(+)Q(A)-Q(B) and P(+)Q(A)Q(B)(- ), and measured as a function of temperature. These measurements provi de information on the time-dependent variation in electrostatic field strength on the Bchl and Bph cofactors. Measurements in the near-infra red absorbance bands have revealed the following. First, the Q(A)(-)Q( B) --> Q(A)Q(B)(-) electron transfer rate is found to be heterogeneous , consisting of at least two distinct kinetic components. At room temp erature, we find a previously unresolved fast kinetic component with a reaction time of 25-40 mu s, depending upon the preparation, that acc ounts for approximately 25% of the total reaction yield. The major com ponent was identified with a reaction time of 210-240 mu s. Below -20 degrees C, Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer shows distrib uted kinetics. The temperature-dependent conversion from biphasic to d istributed kinetics suggests that there is a thermal averaging of conf ormational substates around two reaction center configurations. Intere stingly, direct excitation of the Bph with 532 nm light at low tempera tures appears to alter the electron transfer kinetics, possibly by ind ucing a change in the distribution of conformational states. The react ion kinetics were found to be sensitive to the addition of ethylene gl ycol, which is likely to reflect an osmolarity effect. Second, time-de pendent absorption changes of the Bchl and Bph cofactors are found to be kinetically decoupled. The rapid responses of the Bph bands are int erpreted to reflect electron transfer, while the slower responses of t he Bchl are interpreted to reflect slower relaxation events, possibly including proton uptake. Finally, we find that the electrochromic resp onse and Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer to be sensitive to the preparative state of the reaction center, reflecting differenc es in quinone binding for reaction centers in different states of puri fication.