Kinetic Determination of Cytochrome b₆f Activity In Vitro

While traditional kinetic studies of the cytochrome b₆f complex have frequently relied on measurements within the complex environment of intact leaves or whole-organism systems, such approaches can be limited by overlapping signals and physiological variables. This protocol advances existing frameworks by introducing a streamlined, multi-wavelength spectroscopic approach utilizing a reconstituted in vitro system to elucidate the inter-complex electron transfer kinetics between photosystem I and cytochrome b₆f. Utilizing the JTS-150 pulsed spectrometer, supplied with a Smart Lamp, we monitored the redox transitions of P700⁺ and Cytf by simultaneously measuring the absorbance changes of our isolated complexes system in six different wavelengths (546, 554, 563, 574, 705, and 740 nm). Kinetic analysis was divided into two phases: laser-induced flash kinetics and steady-state actinic induction. We resolved the second-order re-reduction of P700⁺ by plastocyanin, accounting for detector saturation constraints with a 2 ms post-flash delay. Steady-state measurements under actinic light revealed complex Cytf turnover, characterized by a double-exponential decay. Furthermore, dark relaxation kinetics were used to quantify ferredoxin-mediated re-reduction of the cytochrome pool. By allowing the incorporation of specific regulatory and inhibitory factors, this methodology sets the ground for the deconvolution of competing electron pathways. It can therefore be used as a robust framework for assessing the mechanism of regulatory processes on photosynthetic flux. Key features • Activity measurement of isolated photosynthetic complexes. • Assessing interactions between complexes in the photosynthetic apparatus.