The Soret circular dichroism spectrum as a probe for the heme Fe(III)-Met(80) axial bond in horse cytochrome c

A spectroscopic signal sensitive to the strength of the heme iron(III)-Met(80) bond in cytochrome c represents a useful tool that will provide valuable information on the heme pocket region and redox properties of the protein. At present, the 695-nm absorption band is perhaps the simplest diagnostic signal for the axial bond; this band disappears when the Fe(III)-Met(80) bond is disrupted. From the analysis of the Soret region circular dichroism spectrum of cytochrome c under conditions that gradually induce disruption of the Fe(III)-Met(80) bond, we present evidence that the 416-nm spectral dichroic band provides independent information addressing the strength of the axial bond between Fe(III) and Met(80) in cytochrome c. Further, this study demonstrates extension of the diagnostic application to very dilute protein samples based on the useful sample concentration of 5-10 mu M vs 200-300 mu M required for 695-nm absorbance measurements. (C) 1997 Elsevier Science Inc.