Cytochrome bc₁-c_y fusion complexes reveal the distance constraints for functional electron transfer between photosynthesis components

Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH₂): cytochrome (cyt) c oxidoreductases (cyt bc₁ complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt c_y) or a freely diffusible (cyt c₂) electron carrier. Previously, we constructed a functional cyt bc₁-c_y fusion complex that supported Ps growth solely relying on membrane-confined ET (Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta 1757, 346-352). In this work, we further characterized this cyt bc₁-c_y fusion complex, and used its derivatives with shorter cyt c_y linkers as "molecular rulers" to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc₁-c_y fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt c_y linkers exhibited fast, native-like ET rates to the RC via the cyt bc₁. However, shortening the length of the cyt c_y linker decreased drastically this electronic coupling between the cyt bc₁-c_y fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt c_y linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc₁-c_y fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are "hardwired" for cyclic ET.