Spectroscopic and kinetic studies of the tetraheme flavocytochrome c from Shewanella putrefaciens NCIMB400

Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopic studies were carried out on the tetraheme flavocytochrome c from Shewanella. putrefaciens NCIMB400. The EPR spectrum reveals two sets of g-values-g(z) = 2.93, g(y) = 2.28, and g(x) 1.51; and g(z) = 3.58-and the MCD spectrum shows a charge-transfer band at 1510 nm. These data combined show that all four hemes are low spin and have a nitrogenous sixth ligand. Sequence comparisons with other tetraheme cytochromes, particularly that from the purple phototroph H-1-R [Ambler, R. P. (1998) Biochim. Biophys. Acta 1058, 42-47], indicate that the sixth ligands are all histidines. Both the EPR data and the previously reported heme midpoint potentials [-220 and -320 mV; Morris, C. J., Black, A. C., Pealing, S. L., Manson, F. D. C., Chapman, S. K., Reid, G. A., Gibson, D. M., and Ward, F. B. (1994) Biochem. J. 302, 587-593] indicate that the hemes fall into two pairs. Stopped-flow kinetic experiments showed that fumarate-dependent heme oxidation was biphasic (k(cat)[fast] = 400 +/- 20 s(-1); k(cat)[slow] = 34 +/- 3 s(-1)), with each phase having the same amplitude, confirming that the hemes are functionally paired.