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A structural model of Bacillus subtilis cytochrome c-550 has been built based upon hydropathy analysis, sequence alignment, homology modeling, and energy minimization. The model has a single transmembrane α-helix and a water-soluble domain folded around covalently attached heme C. Physical measurements on purified, recombinant cytochrome c-550 have been made to test aspects 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 tryptophans of cytochrome c-550 in the heme domain and the second (i.e., W3) in the transmembrane domain. The indole ring of W115 is within 5 A of the heme macrocycle and is expected to be highly quenched via resonance energy transfer to the heme. In contrast, W3 is at the start of the putative transmembrane helix and could be located a considerable distance from the heme. Forster theory assigns a distance of 42 A from W3 to the heme. This 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 α-helical content of B. subtilis cytochrome c compared to mitochondrial cytochrome c in support of an α-helical transmembrane domain. The ionic strength dependence of redox kinetics for cytochrome c-550 indicates an overall negative charge that is consistent with a calculated p I of 5.4. However, the charge distribution specified by the model indicates a surface for electron exchange that is different from the classical front face used by mitochondrial cytochrome c .