Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers

Abstract

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c 2 and the membrane-anchored cyt c y . In species like R. capsulatus, cyt c y functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c y (S-c y ), by fusing genetically the cyt c 2 signal sequence to the cyt c domain of cyt c y . This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c y regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c y variants at amounts comparable with that of cyt c 2 , and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c y was about 340 mV, similar to that of cyts c 2 or c y . Remarkably, electron transfer kinetics from the cyt bc 1 complex to the photochemical reaction center (RC) mediated by cyt S-c y was distinct from those seen with the cyt c 2 or cyt c y . The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c y interacted with the RC less tightly than cyt c 2 . Comparison of structural models of cyts c 2 and S-c y revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c 2 to the RC are not conserved in cyt c y , whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

“…17,18,22 Subtle differences might be due to the presence of detergents, lipids, or other RC subunits in the purified RC or in the native membrane, and the discrepancies are the same range as observed for the soluble variant of membrane-anchored cytochrome c y . 36 These spectroscopic and electrochemical analyses indicate that the recombinant C-cyt c z has the same protein fold and heme group coordination as in the native cyt c z .…”

Cytochrome bc1-cy Fusion Complexes Reveal the Distance Constraints for Functional Electron Transfer Between Photosynthesis Components

“…These fast rates further evidenced that the monitored ETs were mediated by the membranebound cyts c y , and not by their soluble versions (cyt S-c y ), somehow generated by proteolysis. Otherwise, as described in the accompanying work (38), the ET rates would have become much slower. Sharply contrasting the rates, the amplitudes of ET to the RCs were slightly smaller that the unconnected systems even in the presence of a cyt bc 1 -c y complex with a native cyt c y linker, and became progressively smaller with shorter linkers.…”

“…However, as the membrane-embedded components can diffuse independently from one another within lipid bilayers, differentiating between static "hardwired" electron transport complexes from those undergoing random collisions (17) is difficult to document in Ps membranes (18). In the accompanying work (38), we have demonstrated by creating soluble variants of cyt c y (cyt S-c y ) that this electron carrier needs not be membrane anchored to support Ps growth of R. capsulatus. In this work, we have exploited the functional cyt bc 1 -c y fusion complex that we constructed earlier by fusing genetically cyt c y to the cyt bc 1 complex (19) to probe the physical proximities of the Ps components to one another in R. capsulatus.…”

Cryo-EM Structures of Respiratory bc1-cbb3 type CIII2CIV Supercomplex and Electronic Communication Between the Complexes

“…fraction A-1) were supplemented with either purified full-length cyt c y , or its soluble variant lacking the TMH (i.e., cyt S-c y ) 32 , to yield the bipartite SC+c y and SC+S-c y samples. Following SEC, the elution fractions showed that only the intact cyt c y , but not the cyt S-c y , remained associated with the SC (Fig.…”

“…construction of strains producing bipartite and tripartite fusion SCs. The Δ(petABC::gm) deletion-insertion allele carried by pYO34 32 was transferred using the gene transfer agent (GTA) into the chromosome of R. capsulatus strain MG1 (ccoP::kan) to yield YO12, providing a mutant background lacking both CIII 2 and the CcoP subunit of CIV. A similar strain, M7G-CBC1 that lacks both CIII 2 and the CcoP subunit but overproduces the CcoN and CcoO subunits of CIV 31 , was also used.…”