Heme-peptide models for hemoproteins .1. Solution chemistry of N-acetylmicroperoxidase-8

An improved method for the preparation of the heme octapeptide acetyl-MP8, obtained by proteolysis of horse heart cytochrome c, is described. AcMP8 obeys Beer's law at pH 7.0 in aqueous solution up to a concentration of 3 x 10(-5) M. The self-association constant measured at 25 degrees C (log K-D = 4.04) is an order of magnitude lower than that for MP8, reflecting the role of the N-acetyl protecting group in abolishing intermolecular coordination. However, AcMP8 does form pi-stacked dimers in aqueous solution with increasing ionic strength. A more weakly packed pi-pi dimer reaches a maximum abundance at similar to 3 M ionic strength, but a more tightly packed dimer is favored at mu > 3 M. An equilibrium model based on charge neutralization by specific binding of Na+ ions gives a total molecular charge of 3- for AcMP8 at pH 7.0 and a self-association constant log K-D = 4.20. AcMP8 exhibits six spectroscopically active pH-dependent transitions. The Glu-21 c-terminal carboxylate binds to the heme iron at low pH (pK(a) = 2.1) hut is substituted by His-18 (pK(a) = 3.12) as the pH increases. The two heme propanoic acid substituents ionize with pK(a)'s of 4.95 and 6.1. This is followed by ionization of iron-bound water with a pK(a) = 9.59, Delta H = 48 +/- 1 kJ mol(-1), and Delta S = -22 +/- 3 J K-1 mol(-1). The electronic spectra indicate that AcMP8 is predominantly in the S = 5/2 state at pH 7.0, while the hydroxo complex at pH 10.5 corresponds to an equilibrium mixture of S = 5/2 and S = 1/2 states at 25 degrees C. In the final transition, His-18 ionizes to form the S = 1/2 histidinate complex with a pK(a) of 12.71. AcMP8 is relatively stable under alkaline conditions, dimerizing slowly at high pH (k = 2.59 +/- 0.14 M(-1) s(-1)) to form a high-spin mu-oxo-bridged species. The pH-dependent behavior of AcMP8 in the presence of excess 3-cyanopyridine, however, is markedly different. At low pH, AcMP8 simultaneously bihds the exogenous ligand and the Glu-21 c-terminal carboxylate with a pK(a) < 2. His-18 replaces the carboxylate ligand at higher pH (pK(a) = 2.60), and both heme propanoic acid groups ionize with a mean pK(a) = 5.10. Unlike AcMP8 ... OH-, the axial histidine of the 3-CNPy complex ionizes at near neutral pH (pK(a) = 7.83), prior to being replaced by OH- (pK(a) = 10.13). The sixth transition in the AcMP8/3-CNPy system produces the bis(hydroxo) complex (pK(a) > 13).