Expression and characterization of the diheme cytochrome c subunit of the cytochrome bc complex in Heliobacterium modesticaldum

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 factors of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b6f complex or the monoheme cytochrome c1 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 c4 proteins. The recombinant protein exhibits a single redox midpoint potential of +71 mV versus NHE, which indicates that the two hemes have very similar protein environments.