Structural responses to cavity-creating mutations in an integral membrane protein.

X-ray crystallography has been used to investigate the extent of structural changes in mutants of the purple bacterial reaction center that assemble without a particular ubiquinone or bacteriopheophytin cofactor. In the case of the bacteriopheophytin-exclusion mutant, in which Ala M149 was replaced by Trp (AM149W), the quality of protein crystals was improved over that seen in previous work by minimizing illumination, time, and temperature during the purification protocol and carrying out crystal growth at 4 degrees C after overnight incubation at 18 degrees C. The X-ray crystal structure of the AM149W mutant, determined to a resolution of 2.2 A, showed very little change in protein structure despite the absence of the bacteriopheophytin cofactor. Changes in the electron density map in the region of the cofactor binding site could be accounted for by changes in the conformation of the phytol side chains of adjacent cofactors and the presence of a buried water molecule. Residues lining the vacated binding pocket did not show any significant changes in conformation or increases in disorder as assessed through crystallographic atomic displacement parameters (B-factors). The X-ray crystal structure of a reaction center lacking the primary acceptor ubiquinone through mutation of Ala M248 to Trp (AM248W) was also determined, to a resolution of 2.8 A. Again, despite the absence of an internal cofactor only very minor changes in protein structure were observed. This is in contrast to a previous report on a reaction center lacking this ubiquinone through mutation of Ala M260 to Trp (AM260W) where more extensive changes in structure were apparent. All three mutant reaction centers showed a decrease in thermal stability when housed in the native membrane, but this decrease was smaller for the AM260W mutant than the AM248W complex, possibly due to beneficial effects of the observed changes in protein structure. The lack of major changes in protein structure despite the absence of large internal cofactors is discussed in terms of protein rigidity, the protective influence of the adaptable membrane environment, and the role of small molecules and ions as packing material in the internal cavities created by this type of mutation.

Replacement or exclusion of the B-branch bacteriopheophytin in the purple bacterial reaction centre: the H(B) 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.

On the role of basic residues in adapting the reaction centre-LH1 complex for growth at elevated temperatures in purple bacteria.

The purple photosynthetic bacterium Thermochromatium tepidum is a moderate thermophile, with a growth optimum of 48-50 degrees C. The X-ray crystal structure of the reaction centre from this organism has been determined, and compared with that from mesophilic bacteria such as Blastochloris viridis and Rhodobacter sphaeroides (Nogi T et al. (2000) Proc Natl Acad Sci USA 97: 13561-13566). Structural features that could contribute to the enhanced thermal stability of the Thermochromatium tepidum reaction centre were discussed, including three arginine residues exposed at the periplasmic side of the membrane that are not present in reaction centres from mesophilic organisms, and potentially could increase the affinity of the complex for the surrounding membrane. In the present report these arginine residues, plus a histidine identified from an extensive sequence alignment, were engineered into structurally homologous positions in the Rhodobacter sphaeroides reaction centre, and the effect on the thermal stability of the Rhodobacter sphaeroides complex was examined. We find that these residues do not enhance the thermal stability of the reaction centre, as assessed by absorbance spectroscopy of the bacteriochlorin cofactors in membrane-bound reaction centres. Possible roles of these residues in the Thermochromatium tepidum reaction centre are discussed, and it is proposed that they facilitate stronger binding of the reaction centre to the encircling LH1 antenna complex, through ionic interactions with acidic residues at the C-terminal end of the LH1 alpha-polypeptide. Such an interaction could enhance the stability of the so-called 'RC-LH1 core' complex that is formed between the reaction centre and the LH1 antenna, and which represents the minimal functional photosynthetic unit in all known purple photosynthetic bacteria. Stronger bonding interactions between the two complexes could also contribute to an increase in the rigidity of the photosynthetic membrane in Thermochromatium tepidum, in accord with the general finding that the cytoplasmic membrane from thermophilic eubacteria is less fluid than its counterpart in mesophilic bacteria.