Photochemical trapping of a bacteriopheophytin anion in site‐specific reaction‐center mutants from the photosynthetic bacterium Rhodobacter sphaeroides

Abstract

The mutant YY in the reaction center of Rhodobacter sphaeroides, in which Phel81 on the L chain has been replaced by Tyr, and the double mutant FY, with Tyr210 on the M chain replaced by Phe and Phel81 on the L chain replaced by Tyr, have been constructed by site-directed mutagenesis. The studies described here were performed to complement a previous mutational analysis of mutant FF with Tyr210 replaced by Phe. Both new strains grow photoheterotrophically. The optical absorption spectra of reaction centers isolated from these mutants have band shifts attributable to the monomer bacteriochlorophylls in the vicinity of the substitutions. Photochemical trapping of the bacteriopheophytin anion (I -) indicates that the bacteriopheophytin on the €3 branch is reduced to a much greater extent in FF and FY as compared to YY and wild-type YF. Low temperature (77 K) absorption spectra clearly show that in the wild-type (YF) and YY reaction centers only the 545-nmabsorbing bacteriopheophytin is reduced while in the F F and FY reaction centers both the 535-nm and 545-nm-absorbing bacteriopheophytins are reduced. A simple kinetic analysis is used to explain these results. This analysis suggests that, in order for the observed trapping results to occur, a decrease in the 'cycling' time must take place, that is changes in the rate(s) of charge recombination must accompany the already known decrease in the forward electron transfer rate.The reaction centers from the purple, non-sulfur photosynthetic bacteria Rhodopseudomonas (Rps.) viridis and Rhodohacter (R.) sphaeroides have been crystallized and the X-ray structures solved to atomic resolution (reviewed in Deisenhofer and Michel, 1989;Chang et al., 1986;Tiede et al., 1988;Rees et al., 1989). Biochemical and geneticcomparison with the reaction centers from other photosynthetic bacteria (e.g. R. capsulatus and Chlorojlexus aurantiacus) as well as with photosystem I1 of higher plants indicate that the structures of these complexes are more than likely similar to the already known X-ray structures (Komiyaet al., 1988). The photosynthetic reaction center complex is very symmetric in that the cofactors are arranged with an approximate C2 axis of symmetry which runs between the individual bacteriochlorophylls of the special pair (P) to the iron atom. The result of this symmetry axis is two seemingly equivalent and potential pathways of photoinduced electron transfer Ahhreviutions. P, bacteriochlorophyll dimer; B,, monomer bacteriochlorophyll on the A branch; Bbr monomer bacteriochlorophyll on the B branch; Ha, bacteriopheophytin on the A branch; Hb, bacteriopheophytin on the B branch; Qa, Qb, primary and secondary quinones; I -, reduced bacteriopheophytin; YF, FF, YY and FY refer to the different strains of R. sphueroides with the first letter corresponding to residue 210 on the M chain in the reaction center while the second letter corresponds to residue 181 on the L chain, e.g. Y F is the wild-type protein with Tyr M210 and Phe L181; UQ6, 2,3-dimethoxy-5-methyl-6-(all-tuans)farne...

Low-Temperature Femtosecond-Resolution Transient Absorption Spectroscopy of Large-Scale Symmetry Mutants of Bacterial Reaction Centers

“…This mutation should perturb the environment of the A side cofactors (including B A ), making electron transfer along the A-path less favorable and thus, allowing some B side electron transfer to take place. This suggestion is consistent with the results of Gray et al (1992) who used photopumping methods to generate a steady state population of bacteriopheophytin anion in a single site mutant of Rb. sphaeroides, M210(YfF) (a submutant of sym2-1).…”

Inter- and Intraspecific Variation in Excited-state Triplet Energy Transfer Rates in Reaction Centers of Photosynthetic Bacteria¶

“…1). The discrete asymmetry that the side chains of these residues provide to the RC structure is conserved in all species of photosynthetic bacteria and has been shown to play a significant role in electron and energy transfer functions of the RC (38–50). The residue at the L181 or M208 site can have a significant effect on the redox potentials and absorption spectra of the cofactors with which it can interact—P, B or H (or all)—on its respective side of the RC (26,44,51).…”

Site‐specific mutagenesis of the reaction centre from Rhodobacter sphaeroides studied by Fourier transform Raman spectroscopy: mutations at tyrosine M210 do not affect the electronic structure of the primary donor

“…At low temperature two discrete peaks can be resolved at approximately 545 nm (for the Qx of HL) and 535 nm (for the Qx of HM). The purified mutant reaction centres described in [26] which bore the change Tyr M210--)Phe and Tyr M210-~Leu both exhibited a redshift in the position of the 535 nm band of 2-3 nm, considerably increasing the overlap between the HL and HM Q, peaks [14]. However, in all cases our purified mutant reaction centres showed no alteration in the position of the HM Qx band relative to its position in the spectrum of the wild type, with well resolved peaks being observed at 533 nm and 545 nm for the wild type and at 533 and 546 nm in the Tyr M210~Leu and Tyr M210~Phe mutants.…”

“…sphaeroides which targetted the M210 residue found that replacement of the tyrosine increased the time constant for the reduction of H, from 3.5 ps to 16 ps and 22 ps for changes to Phe and Leu respectively [12], and to 10.5 ps and 16 ps for changes to Phe and Ile [13]. Recently it has also been reported that in reaction centres where the tyrosine at this position has been replaced by a phenylalanine, electron transfer to the Bphe on the inactive branch (H& can be observed under conditions where this does not occur in wild type centres [14], and experiments with mutants at the M2lO/Ll81 positions have also contributed to the proposal that during primary electron transfer the intermediate state P+BM-may be formed [15]. In the latter case the mutations appeared to modulate both the rate and extent of formation of this 'parking state' on the inactive branch.…”