The heme undecapeptide of cytochrome c has been converted to a bis(N-acetylated) derivative by reaction with acetic anhydride. The structure of the product has been confirmed by liquid secondary-ion mass spectrometry. As anticipated, the N-acetylated molecule exhibits much less tendency to aggregate in aqueous solution than its heme undecapeptide precursor. Around neutral pH, one axial ligand on the heme iron is provided by the same histidine residue as in the native cytochrome. The other axial ligand can be varied by the addition of exogenous donor species to produce a range of hemoprotein model compounds exhibiting mixed axial ligation. Contrary to the findings of Othman et al. [Biochemistry 1994, 33, 15437-15448] concerning heme octapeptide, the N-acetylated undecapeptide showed no tendency to bind more than one exogenous ligand per heme. At concentrations approaching millimolar and in the absence of exogenous ligands, the N-acetylated molecule may either be monodispersed, exhibiting a characteristic high-spin (S = (5)/(2)) ferric heme electron paramagnetic resonance (EPR) signal, or exist in an EPR-silent and presumably aggregated form. Interestingly, the system displays a novel dependence on the buffer with regard to which of these two forms is present in a given sample. There is no evidence in any of the spectra for the existence of an intermediate-spin (S = (3)/(2)) ferric heme as suggested by Wang and Van Wart [J. Phys. Chem. 1989, 93, 7925-7931] to be present in aqueous solutions of N-acetylated heme octapeptide. Also, in contrast to another earlier report concerning the underivatized undecapeptide [Clore et al. Inorg. Chim. Acta 1981, 56, 143-148], the N-acetylated molecule showed no evidence of catalase activity. In fact, the heme chromophore was surprisingly unstable in the presence of hydrogen peroxide.
Alkaline forms of the ferric bis(N-acetylated) heme undecapeptide of cytochrome c (N-ac-HUP) and some of its derivatives have been studied by electronic absorption and electron paramagnetic resonance spectroscopies. Surprisingly, even at pH >12, no evidence could be found for the formation of a hydroxyl ion adduct, in direct contrast to a previous report concerning ferric heme peptides encapsulated in detergent micelles (Mazumdar et al. Inorg. Chem. 1991, 30, 700-705). A spectroscopically determined pK(a) of approximately 9 is assigned to the deprotonation of the constituent histidine ligand of heme iron in N-ac-HUP. The present findings are not entirely in keeping with those of an earlier study concerning the properties of N-acetylated heme octapeptide (Wang et al. J. Biol. Chem. 1992, 35, 15310-15318), the differences observed being attributed to the buffering media employed in the two investigations. The implications of the current results in relation to a better understanding of the alkaline transitions observed in hemoglobins and myoglobins is considered.
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