Structural and Functional Study of the Diheme Cytochrome c Subunit of the Cytochrome bc Complex in Heliobacterium modesticaldum.

摘要

This dissertation is focused on the structural and functional study of the unique diheme cytochrome c subunit of the cytochrome bc complex in H. modesticaldum, and its application in determining the stoichiometry between H. modesticaldum's cytochrome bc complex and its reaction center. Heliobacterium modesticaldum is a gram positive, anaerobic, anoxygenic photoheterotrophic bacterium. Its cytochrome bc complex (Rieske/cyt b complex) has some similarities to cytochrome b6f complexes from cyanobacteria and chloroplasts, and also shares some characteristics of typical bacterial cytochrome bc1 complexes. One of the unique features of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b 6f complex or the monoheme cytochrome c 1 in the bc1 complex. To understand the structure and function of this diheme cytochrome c protein, we expressed the N-terminal transmembrane-helix-truncated soluble H. modesticaldum diheme cytochrome c in Escherichia coli. This 25 kDa recombinant protein possesses two c-type hemes, confirmed by mass spectrometry and a variety of biochemical techniques. Sequence analysis of the H. modesticaldum diheme cytochrome c indicates that it may have originated from gene duplication and subsequent gene fusion, as in cytochrome c 4 proteins. The recombinant protein exhibits a single redox midpoint potential of + 71 mV vs NHE, which indicates that the two hemes are in very similar protein binding environments. Further studies by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and ion mobility mass spectrometry (IM-MS) show unambiguously that there is a real structural conformational difference between the oxidized and reduced diheme cytochrome c. A smaller or more compact reduced form might have the function of bringing the two hemes closer to facilitate interheme electron transfer, which also implies a mechanism that is possibly sensitive to the distance between hemes. To determine the stoichiometry of heliobacterial reaction center (RC) and cytochrome bc complex, which is a crucial step toward building a more accurate heliobacterial cyclic electron transfer (CET) model, antibodies against the diheme cytochrome c was raised and quantitative Western blotting was performed to probe and calculate the content of the cytochrome bc complex. The amount of RC was determined by measuring the absorption spectrum of the Bchl g (Bchl g') in the cell methanol extract. A stoichiometry of 14 to 8 RCs per cytochrome bc complex catalytic center was estimated, a much larger number than previously reported, indicating a high turnover number of the cytochrome bc complex.