MINI-MYOGLOBIN - NATIVE-LIKE FOLDING OF THE NO-DERIVATIVE

Mini-myoglobin is a polypeptide fragment (residues 32-139) obtained by limited proteolysis of horse heart apomyoglobin and reconstituted wit h the natural heme. Its functional properties are very similar to thos e of native myoglobin and therefore it may represent a model for testi ng the functional role of the protein fragment encoded by the central exon of myoglobin gene (residues 31-105). Here we have investigated so me properties of the nitric oxide derivative of mini-myoglobin in comp arison with those of NO-myoglobin, to provide more structural informat ion on the heme pocket residues in addition to that already acquired b y electron paramagnetic resonance of the cobalt-substituted mini-myogl obin. At pH 7.0, optical and circular dichroism Soret spectra, as well as electron paramagnetic resonance spectra reveal that the heme orien tation in the pocket and the coordination state of the ferrous iron in NO-mini-myoglobin are similar to those of the whole protein. The spec tra of the NO-mini-myoglobin complex are very sensitive to pH changes at variance to what is observed for the NO-myoglobin derivative in the same pH range (5.5-9.5). In particular, increasing or decreasing pH f rom 7.0, results in a decrease of the extinction coefficient and of th e ellipticity in the Soret region and in a change of the shape of the electron paramagnetic resonance signal. The spectral changes are diagn ostic for a transition from a hexa-coordinated (at pH 7.0) to a penta- coordinated heme (at pH 5.5 or 9.5), with the proximal histidine-iron bond either broken or stretched dramatically. Thus, although mini-myog lobin is able to bind NO in a geometry similar to that of the native p rotein, the resulting NO derivative shows a much higher pH dependence, suggesting that the two lacking side domains are mainly involved in e nhancing the stability of the hemoprotein core.