This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.107.116483v1 94/10/4066    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Mugnol, K. C. U. Articles by Nantes, I. L. PubMed PubMed Citation Articles by Mugnol, K. C. U. Articles by Nantes, I. L.

Spectroscopic, Structural, and Functional Characterization of the Alternative Low-Spin State of Horse Heart Cytochrome c

Katia C. U. Mugnol ^*, Rômulo A. Ando , Rafael Y. Nagayasu ^*, Adelaide Faljoni-Alario , Sergio Brochsztain ^*, Paulo S. Santos , Otaciro R. Nascimento  and Iseli L. Nantes ^*

^* Centro Interdisciplinar de Investigação Bioquímica (CIIB), Prédio I, Universidade de Mogi das Cruzes (UMC), CP 411, Mogi das Cruzes, SP, CEP 08780-911, Brazil; Instituto de Física de São Carlos, Universidade de São Paulo–São Carlos, CP 369, São Carlos, SP, CEP 13560-970, Brazil; Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, São Paulo, CEP 055089-900, Brazil; and Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, CP 26077, São Paulo, CEP 055089-900, Brazil

Correspondence: Address reprint requests to Iseli L. Nantes, Centro Interdisciplinar de Investigação Bioquímica (CIIB), Prédio I, Universidade de Mogi das Cruzes (UMC), Av. Dr. Cândido Xavier Almeida Sousa, 200, Mogi das Cruzes, SP, CEP 08780-911, Brazil. Tel.: 55-11-4798-7103; Fax: 55-11-4798-7102; E-mail: ilnantes{at}umc.br.

The alternative low-spin states of Fe³⁺ and Fe²⁺ 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 Met⁸⁰ by another strong field ligand at the sixth heme iron coordination position, Fe³⁺ ALSScytc exhibited 1-nm Soret band blue shift and enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD 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 because 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 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.