Direct electrometry was used to study the light-induced voltage changes in the Rhodobacter sphaeroides chromatophores adsorbed to a phospholipid-impregnated nitrocellulose film. After the second laser flash, a fast increase in the voltage associated with charge separation was followed by a slower increase attributed to the proton uptake in the QB site of the photosynthetic reaction centers. Kinetics and relative amplitudes of these voltage changes attributed to the QA-.QB-. → QAQBH2 transition, were measured as a function of pH and temperature between +4 and +40 °C. The kinetics can be approximated by a single exponent above +23 °C (100 µs at +25 °C, pH 7.2), whereas below this temperature, it was a good fit of two exponential approximation (65 µs and 360 µs with similar contributions at +10 °C, pH 7.2). The faster component diminished with an apparent pK ∼8.5, whereas the slower one was maintained at a constant level until pH ∼9.5 and then decreased. The calculated activation energy from the temperature dependence of the slower component (55 – 65 kJ/mol) was much higher than that of the faster component (< 10 kJ/mol). The two voltage components can be attributed to the transfer of the first (faster component) and the second (slower component) proton from the reaction center surface to QB. We suggested that higher activation energy of the slower component was due to a conformational change in the reaction center kinetically coupled to the second proton transfer to QBH-.
The faster component diminished in the presence of 1 M KCl, with an apparent pK ∼7.5. To explain this observation, we assume that: (i) the midpoint potential of the QA/QA-. redox pair was higher in 1 M KCl because of the reduced surface potential of chromatophores; (ii) the midpoint potential of the QB-./QBH- redox pair was insensitive to the surface potential change; (iii) the equilibrium constant of the reaction QA-.QB-. ↔ QAQBH- decreased at high ionic strength.