Hemepeptide models for hemoproteins: the behavior of N-acetylmicroperoxidase-11 in aqueous solution.
The acetylation of the hemeundecapeptide prepared by proteolysis of cytochrome c yields a species di(N-acetyl)-microperoxidase-11, NAcMP11, that is monomeric in aqueous solution at least for concentrations below 20 microM, 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 NAcMP11 (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 H2O molecules from the coordination sphere of Fe(III) by the C-terminal Glu-21 carboxylate (giving the axial ligand combination RCOO-/H2O); as the pH is raised, His-18 is deprotonated and coordinates the metal (His/H2O). The next two transitions are due to ionization of heme propionic acid groups; the penultimate is caused by the ionization of Fe(III)-bound H2O (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 NAcMP11 (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 NAcMP11 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 H2O 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.
Marques
H M
HM
Department of Chemistry, University of the Witwatersrand, Johannesburg, South Africa. hmarques@aurum.chem.wits.ac.za
Perry
C B
CB
D004578:Electron Spin Resonance Spectroscopy; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D014867:Water / Q000737:chemistry
D006420:Hemeproteins; D008024:Ligands; D010446:Peptide Fragments; D012996:Solutions; D014867:Water; C121763:N-acetylmicroperoxidase 11; D010544:Peroxidases; C021799:microperoxidase