Articles Effects of Photosynthetic Reaction Center H Protein Domain Mutations on Photosynthetic Properties and Reaction Center Assembly in Rhodobacter sphaeroides † Ali Tehrani, ‡ Roger C. Prince, § and J. Thomas Beatty* ,‡ Department of Microbiology and Immunology, UniVersity of British Columbia, 300-6174 UniVersity BouleVard, VancouVer, BC, Canada, V6T 1Z3, and ExxonMobil Research and Engineering Company, 1545 Route 22 East, Annandale, New Jersey 08801 ReceiVed April 28, 2003; ReVised Manuscript ReceiVed May 27, 2003 ABSTRACT: Purple bacterial photosynthetic reaction center (RC) H proteins comprise three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane R-helix; and a large C-terminal, globular cytoplasmic domain. We studied the roles of these domains in Rhodobacter sphaeroides RC function and assembly, using a mutagenesis approach that included domain swapping with Blastochloris Viridis RC H segments and a periplasmic domain deletion. All mutations that affected photosynthesis reduced the amount of the RC complex. The RC H periplasmic domain is shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RC M. The RC H cytoplasmic domain also functions in RC complex assembly. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RC M is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RC M and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. We suggest that RC M provides a nucleus for RC complex assembly, and that a RC H/M/L assemblage results in a cytoplasmic pool of soluble RC M and RC L proteins to provide precursors for maximal production of the RC complex. Purple phototrophic bacteria have provided a wealth of information on fundamental aspects of electron- and proton- transfer reactions catalyzed by membrane-integral pigment- protein complexes (1, 2). In Rhodobacter sphaeroides, light is absorbed by light-harvesting antennae called LH2 and LH1 and transferred to the reaction center (RC) 1 complex, which operates as a light-driven two-electron/two-proton quinone reductase. Electrons are transferred from a special pair of bacteriochlorophylls (sometimes called D) through the quinone Q A to a second RC-bound quinone called Q B , and protons are translocated from the cytoplasm to Q B to produce a quinol. The Q B quinol leaves the RC and is oxidized at the periplasmic side of the cytochrome b/c 1 complex, resulting in the net translocation of protons from inside to outside the cell membrane, while electrons are transferred from the b/c 1 complex to the RC (3, 4). Thus, a cycle of † This research was supported by a grant from the CIHR to J.T.B. * Address correspondence to the following author. Phone: (604) 822- 6896. Fax: (604) 822-6041. E-mail: jbeatty@interchange.ubc.ca. ‡ University of British Columbia. § ExxonMobil Research and Engineering Company. 1 Abbreviations: BChl, bacteriochlorophyll; D, reaction center special pair of bacteriochlorophylls; LH1, light-harvesting complex 1; LH2, light-harvesting complex 2; QA, reaction center quinone A; QB, reaction center quinone B; RC, reaction center; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. © Copyright 2003 by the American Chemical Society Volume 42, Number 30 August 5, 2003 10.1021/bi0346650 CCC: $25.00 © 2003 American Chemical Society Published on Web 07/10/2003