Identification of amino acid residues in a proton release pathway near the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides.

Insight into control of proton transfer, a crucial attribute of cellular functions, can be gained from investigations of bacterial reaction centers. While the uptake of protons associated with the reduction of the quinone is well characterized, the release of protons associated with the oxidized bacteriochlorophyll dimer has been poorly understood. Optical spectroscopy and proton release/uptake measurements were used to examine the proton release characteristics of twelve mutant reaction centers, each containing a change in an amino acid residue near the bacteriochlorophyll dimer. The mutant reaction centers had optical spectra similar to wild-type and were capable of transferring electrons to the quinones after light excitation of the bacteriochlorophyll dimer. They exhibited a large range in the extent of proton release and in the slow recovery of the optical signal for the oxidized dimer upon continuous illumination. Key roles were indicated for six amino acid residues, Thr L130, Asp L155, Ser L244, Arg M164, Ser M190, and His M193. Analysis of the results points to a hydrogen-bond network that contains these residues, with several additional residues and bound water molecules, forming a proton transfer pathway. In addition to proton transfer, the properties of the pathway are proposed to be responsible for the very slow charge recombination kinetics observed after continuous illumination. The characteristics of this pathway are compared to proton transfer pathways near the secondary quinone as well as those found in photosystem II and cytochrome c oxidase.

A bound iron porphyrin is redox active in hybrid bacterial reaction centers modified to possess a four-helix bundle domain.

In this paper we report the design of hybrid reaction centers with a novel redox-active cofactor. Reaction centers perform the primary photochemistry of photosynthesis, namely the light-induced transfer of an electron from the bacteriochlorophyll dimer to a series of electron acceptors. Hybrid complexes were created by the fusion of an artificial four-helix bundle to the M-subunit of the reaction center. Despite the large modification, optical spectra show that the purified hybrid reaction centers assemble as active complexes that retain the characteristic cofactor absorption peaks and are capable of light-induced charge separation. The four-helix bundle could bind iron-protoporphyrin in either a reduced and oxidized state. After binding iron-protoporphyrin to the hybrid reaction centers, light excitation results in a new derivative signal with a maximum at 402 nm and minimum at 429 nm. This signal increases in amplitude with longer light durations and persists in the dark. No signal is observed when iron-protoporphyrin is added to reaction centers without the four-helix bundle domain or when a redox-inactive zinc-protoporphyrin is bound. The results are consistent with the signal arising from a new redox reaction, electron transfer from the iron-protoporphyrin to the oxidized bacteriochlorophyll dimer. These outcomes demonstrate the feasibility of binding porphyrins to the hybrid reaction centers to gain new light-driven functions.