Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Substitution of isoleucine L177 by histidine in Rhodobacter sphaeroides reaction center results in the covalent binding of PA bacteriochlorophyll to the L subunit

In this work, we report the unique case of bacteriochlorophyll (BChl) - protein covalent attachment in a photosynthetic membrane complex caused by a single mutation. The isoleucine L177 was substituted by histidine in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Pigment analysis revealed that one BChl molecule was missing in the acetone-methanol extract of the I(L177)H RCs. SDS-PAGE demonstrated that this BChl molecule could not be extracted with organic solvents apparently because of its stable covalent attachment to the mutant RC L-subunit. Our data indicate that the attached bacteriochlorophyll is one of the special pair BChls, P(A). The chemical nature of this covalent interaction remains to be identified.     

Organization in photosynthetic membranes of purple bacteria in vivo: The role of carotenoids

Photosynthesis in purple bacteria is performed by pigment–protein complexes that are closely packed within specialized intracytoplasmic membranes. Here we report on the influence of carotenoid composition on the organization of RC–LH1 pigment–protein complexes in intact membranes and cells of Rhodobacter sphaeroides. Mostly dimeric RC–LH1 complexes could be isolated from strains expressing native brown carotenoids when grown under illuminated/anaerobic conditions, or from strains expressing green carotenoids when grown under either illuminated/anaerobic or dark/semiaerobic conditions. However, mostly monomeric RC–LH1 complexes were isolated from strains expressing the native photoprotective red carotenoid spheroidenone, which is synthesized during phototrophic growth in the presence of oxygen. Despite this marked difference, linear dichroism (LD) and light-minus-dark LD spectra of oriented intact intracytoplasmic membranes indicated that RC–LH1 complexes are always assembled in ordered arrays, irrespective of variations in the relative amounts of isolated dimeric and monomeric RC–LH1 complexes. We propose that part of the photoprotective response to the presence of oxygen mediated by synthesis of spheroidenone may be a switch of the structure of the RC–LH1 complex from dimers to monomers, but that these monomers are still organized into the photosynthetic membrane in ordered arrays. When levels of the dimeric RC–LH1 complex were very high, and in the absence of LH2, LD and ?LD spectra from intact cells indicated an ordered arrangement of RC–LH1 complexes. Such a degree of ordering implies the presence of highly elongated, tubular membranes with dimensions requiring orientation along the length of the cell and in a proportion larger than previously observed.     

Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the H_A pocket of the RC. The presence of a 4-coordinate Zn^2 + ion in the H_A bacteriochlorin instead of BPhe results in a small decrease in the rates of the P* → P⁺H_A⁻ → P⁺Q_A⁻ electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn^2 + by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the H_A Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the H_B pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at H_A in both BChl- and Zn-BChl-containing RCs found in nature.     

Kinetic analysis of demetalation of bacteriochlorophyll c and e homologs purified from green sulfur photosynthetic bacteria

Substituent-dependent demetalation kinetics of natural bacteriochlorophyll (BChl) c and e homologs purified from two green sulfur photosynthetic bacteria was first studied. Separated BChl e homologs, which possessed a formyl group at the 7-position of their chlorin macrocycles, exhibited a significantly slow removal of central magnesium to free-base bacteriopheophytins in acidic aqueous acetone compared with the corresponding BChl c homologs, which possessed a methyl group at the 7-position. Additional methyl groups at the 8(2)-position of both BChl c and e molecules had little effect on the demetalation kinetics.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Replacement or exclusion of the B-branch bacteriopheophytin in the purple bacterial reaction centre: The HB cofactor is not required for assembly or core function of the Rhodobacter sphaeroides complex

All of the membrane-embedded cofactors of the purple bacterial reaction centre have well-defined functional or structural roles, with the exception of the bacteriopheophytin (H_B) located approximately half-way across the membrane on the so-called inactive- or B-branch of cofactors. Sequence alignments indicate that this bacteriochlorin cofactor is a conserved feature of purple bacterial reaction centres, and a pheophytin is also found at this position in the Photosystem-II reaction centre. Possible structural or functional consequences of replacing the H_B bacteriopheophytin by bacteriochlorophyll were investigated in the Rhodobacter sphaeroides reaction centre through mutagenesis of residue Leu L185 to His (LL185H). Results from absorbance spectroscopy indicated that the LL185H mutant assembled with a bacteriochlorophyll at the H_B position, but this did not affect the capacity of the reaction centre to support photosynthetic growth, or change the kinetics of charge separation along the A-branch of cofactors. It was also found that mutation of residue Ala M149 to Trp (AM149W) caused the reaction centre to assemble without an H_B bacteriochlorin, demonstrating that this cofactor is not required for correct assembly of the reaction centre. The absence of a cofactor at this position did not affect the capacity of the reaction centre to support photosynthetic growth, or the kinetics of A-branch electron transfer. A combination of X-ray crystallography and FTIR difference spectroscopy confirmed that the H_B cofactor was absent in the AM149W mutant, and that this had not produced any significant disturbance of the adjacent ubiquinol reductase (Q_B) site. The data are discussed with respect to possible functional roles of the H_B bacteriopheophytin, and we conclude that the reason(s) for conservation of a bacteriopheophytin cofactor at this position in purple bacterial reaction centres are likely to be different from those underlying conservation of a pheophytin at the analogous position in Photosystem-II.