Hemepeptide models for hemoproteins: the behavior of N-acetylmicroperoxidase-11 in aqueous solution

Abstract

The acetylation of the hemeundecapeptide prepared by proteolysis of cytochrome c yields a species di ( N-acetyl)-microperoxidase-ll, NAcMP11, that is monomeric in aqueous solution at least for concentrations below 20 μM, in contrast to MP11 itself, which aggregates because of intermolecular coordination of Fe (III) by the N-terminal amino group or the amino group of the side chain of Lys-13. The present report complements a report by Peterson and co-workers on the preparation and properties of NAcMPl 1 (Inorg. Chem. 35 (1996) 6885). We show that NAcMP11 has six spectroscopically observable pH-dependent transitions at 1.90±0.03, 3.37±0.01, 4.6±0.1, 5.4±0.03, 9.56±0.01 and 12.4±0.03. The first is probably due to displacement of one of two H 2O molecules from the coordination sphere of Fe(III) by the C-terminal Glu-21 carboxylate (giving the axial ligand combination RCOO −/H 2O); as the pH is raised, His-18 is deprotonated and coordinates the metal (His/H 2O). The next two transitions are due to ionization of heme propionic acid groups; the penultimate is caused by the ionization of Fe(III)-bound H 2O (His/OH −); and the final transition is from ionization of His-18 to form a histidinate (His −/OH −). The EPR spectrum of NAcMP11 at pH 0.7 is consistent with a mixture of a di(aqua) and a mono(aqua) species. Both the aqua complex of NAcMPll (at pH 7.6) and the hydroxo complex (at pH 11.0) are in equilibrium between a quantum-mechanically admixed spin state ( S = 3/2, 5/2) and a low-spin state ( S= 1/2). The crystal field parameters of the two complexes (which are similar) as derived from the EPR spectrum are reported. The EPR spectrum at pH 13.8 shows that the hydroxo-histidinate complex of NAcMPl 1 undergoes a slow reaction, possibly to form a di(hydroxo) complex with displacement of the histidinate ligand, or a dimerization with the histidinate acting as bridging ligand. The coordinated H 2O molecule in NAcMP11 is readily replaced by an exogenous ligand, and binding constants for coordination of cyanide, imidazole, azide and chloride are reported. NAcMP-11 is shown to display similar physical and chemical properties to the analogous octapeptide, NAcMP-8, but is easier to prepare; this makes NAcMP-11 a useful alternative model for the hemoproteins.