Characterization of a structural model of membrane bound cytochrome c-550 from Bacillus subtilis

A structural model of Bacillus subtilis cytochrome c-550 has been built bas ed upon hydropathy analysis, sequence alignment, homology modeling, and ene rgy minimization. The model has a single transmembrane alpha-helix and a wa ter-soluble domain folded around covalently attached heme C. Physical measu rements on purified, recombinant cytochrome c-550 have been made to test as pects of the model. Excitation at either 280 or 295 nm yields fluorescence with an emission maximum at 334 nm and a quantum yield of 25% relative to n -acetyltryptophanamide. The model places one (i.e., W115) of the two trypto phans of cytochrome c-550 in the heme domain and the second (i.e., W3) in t he transmembrane domain. The indole ring of W115 is within 5 Angstrom of th e heme macrocycle and is expected to be highly quenched via resonance energ y transfer to the heme. In contrast, W3 is at the start of the putative tra nsmembrane helix and could be located a considerable distance from the heme . Forster theory assigns a distance of 42 Angstrom from W3 to the heme. Thi s distance is important in adjusting the relative positions of the membrane -spanning and heme-binding domains. Circular dichroism measurements in the ultraviolet region indicate increased alpha-helical content of B. subtilis cytochrome c compared to mitochondrial cytochrome c in support of an alpha- helical transmembrane domain. The ionic strength dependence of redox kineti cs for cytochrome c-550 indicates an overall negative charge that is consis tent with a calculated pI of 5.4. However, the charge distribution specifie d by the model indicates a surface for electron exchange that is different from the classical front face used by mitochondrial cytochrome c. (C) 2000 Academic Press.