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

Sara L Pealing , Myles R Cheesman, Graeme A Reid, Andrew J Thomson , F Bruce Ward , Stephen K Chapman

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    Abstract

    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.

    Original languageEnglish
    Pages (from-to)6153-6158
    Number of pages6
    JournalBiochemistry
    Volume34
    Issue number18
    DOIs
    Publication statusPublished - May 1995

    Keywords

    • ENZYME
    • SEQUENCE
    • RESOLUTION
    • IRON(III)
    • ELECTRON-TRANSFER
    • HEMOPROTEINS
    • CYTOCHROME-C3
    • MAGNETIC CIRCULAR-DICHROISM
    • FUMARATE REDUCTASE
    • SUCCINATE

    Cite this

    Pealing , S. L., Cheesman, M. R., Reid, G. A., Thomson , A. J., Ward , F. B., & Chapman, S. K. (1995). Spectroscopic and kinetic studies of the tetraheme flavocytochrome c from Shewanella putrefaciens NCIMB400. Biochemistry, 34(18), 6153-6158. https://doi.org/10.1021/bi00018a018