Spectroscopic, structural and functional characterization of the alternative low spin state of horse heart cytochrome C

• Grant number: 08/02933-6
• Support Opportunities: Regular Research Grants - Publications - Scientific article
• Start date: May 01, 2008
• End date: April 30, 2009
• Field of knowledge: Biological Sciences - Biophysics - Radiology and Photobiology
• Principal Investigator: Iseli Lourenço Nantes Cardoso
• Grantee: Iseli Lourenço Nantes Cardoso
• Host Institution: Pró-Reitoria de Pesquisa, Pós-Graduação e Extensão e Assuntos Comunitários. Universidade de Mogi das Cruzes (UMC). Mogi das Cruzes , SP, Brazil

Abstract

The alternative low spin states of Fe3+ and Fe2+ cytochrome c (ALSScytc) induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance (EPR), UV-visible, circular dichroism (CD), magnetic circular dichroism (MCD), fluorescence and Raman Resonance (RR). Consistent with the replacement of Met80 by another strong field ligand at the sixth heme iron coordination position, Fe3+ ALSScytc exhibited 1 nm Soret band blue shift and e enhancement accompanied by disappearance of the 695 nm charge transfer band. The RR, CD and MCD spectra of Fe3+ and Fe2+ ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold since the Trp59 fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His33 and His26 carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His33 replaced by Asn) pointing out Lys79 as the probable heme iron ligand. Fe3+ ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential around 200 millivolts lower than the wild-type protein (+ 220 mV) and was more susceptible to the attack of free radicals. (AU)