Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome C.
The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) 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 approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.
Mugnol
Katia C U
KC
Centro Interdisciplinar de Investigação Bioquímica, Prédio I, Universidade de Mogi das Cruzes, CP 411, Mogi das Cruzes, SP, CEP 08780-911, Brazil.
Ando
Rômulo A
RA
Nagayasu
Rafael Y
RY
Faljoni-Alario
Adelaide
A
Brochsztain
Sergio
S
Santos
Paulo S
PS
Nascimento
Otaciro R
OR
Nantes
Iseli L
IL
D000818:Animals; D003198:Computer Simulation; D045304:Cytochromes c / Q000378:metabolism* / Q000648:ultrastructure*; D006736:Horses / Q000378:metabolism*; D007501:Iron / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods*; D013113:Spin Labels
D013113:Spin Labels; D045304:Cytochromes c; D007501:Iron