Heme and cysteine microenvironments of tuna apomyoglobin. Evidence of two independent unfolding regions.

Abstract

The heme and cysteine microenvironments of bluefin tuna apomyoglobin have been investigated by examining the fluorescence properties of two extrinsic chromophores, i.e., ANS and 1,5-AEDANS. 1,5-AEDANS was covalently bound to the single cysteine residue found in the primary structure of tuna apomyoglobin. Recombination experiments with hemin showed that tuna apomyoglobin does not bind 1,5-AEDANS in the same binding site of the heme, although the fluorescence properties of the covalently bound 1,5-AEDANS strongly suggest that the dye is embedded in a rather nonpolar microenvironment. ANS was selected because of its ability to bind the apomyoglobin in the same nonpolar moiety of the heme. Acidification of apoMb--AEDANS to pH 3.0 produced an increase of 1,5-AEDANS fluorescence intensity and a shift of its emission maximum from 475 to 470 nm. In the same pH range apomyoglobin lost its ability to bind ANS. Two independent transitions were observed with increasing concentrations of guanidine. Low guanidine concentration (less than 1.0 M) unfolded the heme binding site as indicated by the disappearance of ANS fluorescence, whereas higher denaturant concentration was required to produce full normalization of 1,5-AEDANS emission spectrum.