Primary structure of the M subunit of the reaction center from Rhodopseudomonas sphaeroides

Abstract

The reaction center is a membrane-bound bacteriochlorophyll-protein complex that mediates the primary photochemical events in the photosynthetic bacterium Rhodopseudomoas sphaeroides. The previously determined amino-terminal sequences of the three subunits of the reaction center protein were used to design synthetic mixed oligonucleotide probes for the structural genes encoding the subunits. One of these probes was used to isolate and clone a fragment of DNA from R. aphaeroides that contained the gene encoding the M subunit. The nucleotide sequence of this gene was determined by the dideoxy method. In addition, a number of tryptic and chymotryptic peptides from the M protein were isolated and subjected to sequence analysis, and the sequence of the carboxyl terminus was determined. Together with the amino-terminal sequence, the data establish the primary structure of the M protein. The distribution of hydrophobic residues in the amino acid sequence suggests the presence of five membrane-spanning segments. A significant homology was found between the amino acid sequence of the M subunit and a thylakoid membrane protein (Mr 32,000) from spinach that has been implicated in herbicide and quinone binding.The reaction center (RC) is the site of the initial photochemical charge separation in the electron transfer process of photosynthesis (for reviews, see refs. 1 and 2). In the purple bacterium Rhodopseudomnas sphaeroides, the RC is part of the photosynthetic apparatus found in the highly invaginated cytoplasmic membrane that is synthesized by the bacterium under anaerobic conditions (3). The RC protein (Mr 100,000) contains three highly hydrophobic subunits, designated L, M, and H, in a 1:1:1 stoichiometry (4, 5). The cofactors found in the RC are four bacteriochlorophylls, two bacteriopheophytins, two ubiquinones, and one iron.The determination of the amino acid sequence of the RC polypeptides is essential to the detailed elucidation of the RC structure. The sequences of 25-28 amino-terminal residues of each subunit have been determined previously by automated Edman degradation (6). These were used to design mixed-sequence oligonucleotide hybridization probes for the structural genes of each of the subunits. The probe for the gene encoding the M subunit was used to identify a restriction fragment of R. sphaeroides DNA that contained the gene. The nucleotide sequence of this gene was determined by the dideoxy method and is presented together with the amino acid sequence derived from it. The sequences of the carboxyl terminus and of several peptides were determined for corroboration of the sequence obtained from the DNA. Preliminary accounts of this work have been given (7,8).EXPERIMENTAL PROCEDURES Materials. Large fragment Escherichia coli DNA polymerase I, M13mp7 replicative form (RF) DNA, M13 24-base-pair primer fragment, dideoxynucleotides, and the restriction enzymes Acc I, BamHI, EcoRI, HindIII, Hpa II, Kpn I, Sal I, Sma I, and Taq I were obtained from Bethesda Research Laboratories. T4 polynucleotide k...

Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides

“…capsulatus, Rb. sphaeroides, Rhodospirillum rubrum and Rhodopseudomonas viridis (Williams et al 1986). The symmetry-related amino acid in the L polypeptide is phenylalanine at position L181.…”

My Road to Biophysics: Picking Flowers on the Way to Photosynthesis

“…The sequence of the H-subunit showed only one transmembrane helix (157). An exciting result of the sequence work was the homology that was found between the L-and M-subunits and the D 1 and D 2 polypeptides found in green plants (159).…”

“…These sequences were later used by JoAnn Williams, a talented graduate student with a green thumb for recombinant DNA techniques. JoAnn isolated the genes that encode the L-and M-subunits and determined the sequence of these subunits (158,159). Each subunit showed five hydrophobic regions that were postulated to form transmembrane helices.…”

Tansley Review No. 109.

“…This RC consists of three polypeptides, named H (heavy), M (medium) and L (light) after their mobility on SDS-polyacrylamide gels (Feher & Okamura, 1978). Their actual molecular masses are : L, 31 319 Da ; M, 34 265 Da ; H, 28 534 Da (Williams et al, 1984(Williams et al, , 1986. Subunits L and M each contain five membrane-spanning α-helices.…”