11 - Reactivity of Recombinant Cytoglobin with Peroxides and Amyloid Structure in Peroxide-Activated Cytoglobin

Abstract

Introduction Cytoglobin (Cygb) is a member of the globin superfamily, and it is expressed at different levels in many tissues, such as neurons, fibroblasts and connective tissue. The biological role played by Cygb is not well established and different functions have been postulated. In Cygb the heme group presents different coordination when compared with the well-known hemoglobin (Hb) and myoglobin (Mb) with implications for the protein potential and function. Cygb is a hexacoordinated globin present in humans and other vertebrates in which the heme iron sixth coordination position of heme iron in the ferric and ferrous form is occupied by a histidine residue. Considering the presence of a bis-histidine hexacoordinated heme iron, it was expected that, differently of Mb that is pentacoordinated and cytochrome c (Cytc) that is hexacoordinated but with a labile sixth ligand (Met80), Cygb might not be able to react with peroxides. Objective: In the present study it was investigated the reactivity of Cygb with hydrogen peroxide (HOOH), tert-butylhydroperoxide (t-BuOOH) and cumene hydroperoxide (CuOOH). Results Cygb was assayed with different concentrations of peroxides and spectra were run at different times. The ratio of Soret band intensity at 406 nm and 421 nm was determined at different times and the initial rate of the reaction plotted versus peroxide concentrations. The catalytic constants k cat /K M for CuOOH, t-BuOOH and HOOH were 0.0028 ± 0.0006, 0.0032 ± 0,0006, 0.0061± 0,0010 s -1 .µM -1 , respectively. The reactivity was corroborated by EPR measurements, that it has demonstrated that was generated one oxidant equivalent in the heme iron (Fe 4+ =O) and the other at a tyrosine residue (tyrosyl radical). The generation of free radicals during the catalytic cycle of Cygb led to the formation of amyloid fibril structures organized into networks and featured like leaves of ferns detected by scanning electron microscopy. Conclusion These studies provide new and important contributions to the multiple biological roles that can be played by globin including repercussions on cellular signaling processes related to oxidative stress and degenerative diseases.