Examination of stability of mutant photosynthetic reaction center of Rhodobacter sphaeroides I(L177)H and determination of location of bacteriochlorophyll covalently bound to the protein
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We demonstrated earlier that as a result of the I(L177)H mutation in the photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides, one of the bacteriochlorophylls (BChl) binds with the L-subunit, simultaneously raising coordination stability of the central magnesium atom of the bacteriochlorophyll associated with the protein. In this study, spectral properties of wild type RC and I(L177)H in the presence of urea and SDS as well as at 48°C were examined. It is shown that the I(L177)H mutation decreases the RC stability. Under denaturing conditions, some changes indicating breakdown of oligomeric structure of the complex and loss of interaction between pigments and their protein environment are observed in I(L177)H RC spectra. In addition, pheophytinization of bacteriochlorophylls occurs in both types of RC in the presence of SDS. However, an 811-nm band is observed in the spectrum of the mutant RC under these conditions, which indicates retention of one of the BChl molecules in the protein binding site and stable coordination of its central magnesium atom. It is shown that in both types of RC, monomeric BChl BB can be modified by sodium borohydride treatment and then extracted by acetone-methanol mixture. Spectral properties of the BChl covalently bound with the protein in I(L177)H RC do not change. The results demonstrate that BChl PA is the molecule of BChl tightly bound with the L-sub- unit in mutant RC as it was supposed earlier.
Key wordsbacterial photosynthesis site-directed mutagenesis coordination of magnesium atom reaction center bacteriochlorophyll Rhodobacter sphaeroides stability of membrane-associated proteins
special pair of bacteriochlorophylls
- PA and BA
bacteriochlorophylls of active electron transport branch
- PB and BB
bacteriochlorophylls of inactive electron transfer branch
sodium dodecyl sulfate
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