The rate of the photocycle (quinone reduction cycle) was measured under continuous light excitation in an isolated reaction center protein of the photosynthetic bacterium Rhodobacter sphaeroides. The rate is determined by the slowest step of the photocycle, which could be the photochemistry (charge separation), the quinone/quinol and cytochrome c2+/c3+ exchanges, or proton delivery to the secondary quinone. The photocycle was driven by high light intensity of a laser diode (5 W/cm2 at 808 nm) to avoid light limitation of the observed rate. The fast turnover of the reaction center (up to 103 s-1) was slowed down by inhibition of the proton delivery to the secondary quinone by transition metal ions (Cd2+ and Ni2+), by mutation of a key protonatable group (L213Asp → Asn), or by use of low-affinity ubiquinone (UQ 0) to the secondary quinone binding site. Although in all of these cases the rate of turnover was 2-3 orders of magnitude less than that of the primary photochemistry marked light intensity dependence was observed. The rate of the photocycle increased from 7 s-1 (Ni2+, low light intensity) to 27 s-1 (high light intensity) at pH 8.4. The anomalous reacceleration is due to alternative events on the acceptor side induced by continuous excitation. We argue that the continuous excitation of the protein trapped in the reduced acceptor (QA-QB -) state produces short-lived reduced bacteriopheophytin (1 -) that delivers activation energy to anomalous changes on the acceptor side as second interquinone electron transfer before proton uptake or increase of the quinone dissociation constant.
ASJC Scopus subject areas
- Organic Chemistry