The role of betaArg-10 in the B800 bacteriochlorophyll and carotenoid pigment environment within the light-harvesting LH2 complex of Rhodobacter sphaeroides.
Previous work has suggested that the betaArg-10 residue forms part of the binding site for the B800 bacteriochlorophyll in the LH2 complex of Rhodobactersphaeroides [Crielaard, W., Visschers, R. W., Fowler, G. J. S., van Grondelle, R., Hellingwerf, K. J., Hunter, C. N. (1994) Biochim. Biophys. Acta1183, 473-482], and this is consistent with the X-ray crystallographic data that have been subsequently obtained for the related LH2 complex from Rhodopseudomonas acidophila [McDermott, G., Prince, S. M., Freer, A. A., Hawthornthwaite-Lawless, A. M., Papiz, M. Z., Cogdell, R. J., Isaacs, N. W. (1995) Nature 374, 517-521]. Therefore, in order obtain more information about the B800 binding site and its effect on the B800 absorption band, betaArg-10 was replaced by residues Met, His, Asn, Leu, and Lys (in addition to the Glu mutant described in our previous work); these residues were thought to represent a suitable range of amino acid shape, charge, and hydrogen-bonding ability. This new series of betaArg-10 mutants, in the form of LH2 complexes in the native membrane, has been characterized using a variety of biochemical and spectroscopic techniques in order to determine the ways in which the mutants differ from wild-type (WT) LH2. For example, most of the mutant LH2 complexes were found to have blue-shifted B800 absorption bands ranging from 794 to 783 nm at 77 K; the exception to this trend is the betaArg-10 to Met mutant, which absorbs maximally at 798 nm. These blue shifts decrease the spectral overlap between the "B800" and B850 pigments, which allowed us to examine the nature of the B800 to B850 transfer step for the betaArg-10 mutant LH2 complexes by carrying out a series of room temperature subpicosecond energy transfer measurements. The results of these measurements demonstrated that the reduced overlap leads to a slower B800 to B850 transfer, although the alterations at betaArg-10 were found to have little effect on the efficiency of internal energy transfer within LH2. Similarly, carotenoid to bacteriochlorophyll energy transfer was largely unaffected, although shifts in the excitation spectra in the carotenoid region were noted. These betaArg-10 mutant complexes provide an opportunity to investigate the structural requirements for the binding of monomeric bacteriochlorophyll and to examine the basis of the red shift seen for bacteriochlorophyll in photosynthetic complexes, in addition to providing new information about the environment of the carotenoid pigments in this complex.
Fowler
G J
GJ
Robert Hill Institute for Photosynthesis and Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K. g.fowler@sheffiedl.ac.uk
Hess
S
S
Pullerits
T
T
Sundström
V
V
Hunter
C N
CN
D001120:Arginine / Q000737:chemistry*; D001426:Bacterial Proteins; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004735:Energy Transfer; D045342:Light-Harvesting Protein Complexes; D008956:Models, Chemical; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000737:chemistry*; D013054:Spectrophotometry, Atomic
C081521:B800-850 light-harvesting complex, bacteria; D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D001120:Arginine