EFFECT OF TEMPERATURE AND SURFACE-POTENTIAL ON THE ELECTROGENIC PROTON UPTAKE IN THE Q(B) SITE OF THE RHODOBACTER-SPHAEROIDES PHOTOSYNTHETIC REACTION-CENTER - Q(A)(-CENTER-DOT)Q(B)(-CENTER-DOT)-]Q(A)Q(B)H(2) TRANSITION

Direct electrometry was used to study the light-induced voltage change s in the Rhodobacter sphaeroides chromatophores adsorbed to a phosphol ipid-impregnated nitrocellulose film. After the second laser flash, a fast increase in the voltage associated with charge separation was fol lowed by a slower increase attributed to the proton uptake in the QB s ite of the photosynthetic reaction centers. Kinetics and relative ampl itudes of these voltage changes attributed to the Q(A)(-.)Q(B)(-.) --> Q(A)Q(B)H(2) transition, were measured as a function of pH and temper ature between +4 and +40 degrees C. The kinetics can be approximated b y a single exponent above +23 degrees C (100 mu s at +25 degrees C, pH 7.2), whereas below this temperature, it was a good fit of two expone ntial approximation (65 mu s and 360 mu s with similar contributions a t +10 degrees C, pH 7.2). The faster component diminished with an appa rent pK similar to 8.5, whereas the slower one was maintained at a con stant level until pH similar to 9.5 and then decreased. The calculated activation energy from the temperature dependence of the slower compo nent (55 - 65 kJ/mol) was much higher than that of the faster componen t (< 10 kJ/mol). The two voltage components can be attributed to the t ransfer of the first (faster component) and the second (slower compone nt) proton from the reaction center surface to QB. We suggested that h igher activation energy of the slower component was due to a conformat ional change in the reaction center kinetically coupled to the second proton transfer to Q(B)H(-). The faster component diminished in the pr esence of 1 M KCl, with an apparent pK similar to 7.5. To explain this observation, we assume that: (i) the midpoint potential of the Q(A)/Q (A)(-.) redox pair was higher in 1 M KCl because of the reduced surfac e potential of chromatophores; (ii) the midpoint potential of the Q(B) (-.)/Q(B)H(-) redox pair was insensitive to the surface potential chan ge; (iii) the equilibrium constant of the reaction Q(A)(-.)Q(B)(-.) <- > Q(A)Q(B)H(-) decreased at high ionic strength.