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--gz = 2.93, gy = 2.28, and gx = 1.51; and gz = 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. (1991) Biochem. 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., & 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 (kcat[fast] = 400 +/- 20 s-1; kcat[slow] = 34 +/- 3 s-1), with each phase having the same amplitude, confirming that the hemes are functionally paired.