Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria

Abstract

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivaxgelatinosus and Rhodobactersphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria.

Roadmap of electrons from donor side to the reaction center dimer of photosynthetic purple bacteria with mutated cytochromes

“…sphaeroides strain 2.4.1 (cycA) and Rvx. gelatinosus (pufC) were deleted as described earlier [Kis et a. 2022].…”

“…Myxothiazol and terbutryn were routinely used in 10 μM and 100 μM concentrations to inhibit the cyclic electron transfer through the cytochrome bc1 complex and the QA -→ QB interquinone electron transfer in the RC, respectively. To oxidize the periplasmic electron donors of pufC, 1 mM potassium ferricyanide was added to the sample [Yoshida et al 1999;Kis et al 2022]. Ferrocene was used as external electron donor to P + .…”

“…gelatinosus consists of several electron donors (HiPIP and cytochrome c8) under our growing conditions (anaerobicity in the light), it is recommended to track the oxidationreduction reactions by detection of the re-reduction of P + rather than the oxidation of the electron donors. While the cytochromes have characteristic intense and sharp absorption band around 550 nm [Kis et al 2022], the absorption bands of HiPIP are broad and of low intensity around 480 nm [Bartsch 1971]. Consequently, there is no way to detect the oxidation of HiPIP in the presence of highly absorbing cytochromes in intact cells.…”

Roadmap of electrons from donor side to the reaction center of photosynthetic purple bacteria with mutated cytochromes

“…Myxothiazol and terbutryn were routinely used in 10 μM and 100 μM concentrations, respectively, to inhibit the cyclic electron transfer through the cytochrome bc 1 complex and the Q A ‒ → Q B inter-quinone electron transfer in the RC. To oxidize the periplasmic electron donors of pufC , 1 mM potassium ferricyanide was added to the sample [Yoshida et al 1999 ; Kis et al 2022 ]. Ferrocene was used as external electron donor to P + .…”

“…gelatinosus consists of several electron donors (HiPIP and cytochrome c 8 ) under our growing conditions (anaerobicity in the light), it is recommended to track the oxidation–reduction reactions by detection of the re-reduction of P + rather than the oxidation of the electron donors. While the cytochromes have characteristic intense and sharp absorption band around 550 nm [Kis et al 2022 ], the absorption bands of HiPIP are broad and of low intensity around 480 nm [Bartsch 1971 ]. Consequently, there is no way to detect the oxidation of HiPIP in the presence of highly absorbing cytochromes in intact cells.…”