Haempeptide models for haemoproteins Part 3 N-Acetylmicroperoxidase-8: EPR, Mössbauer and magnetic susceptibility studies on an iron(III) porphyrin in thermal equilibrium between S = 3/2, 5/2 and S = 1/2 states

Abstract

The N-terminus acetylated haemoctapeptide from cytochrome c, acetylmicroperoxidase-8 (AcMP8·OH₂) displays complex magnetic behaviour, contingent on its pH-dependent axial ligand combinations (histidine, H₂O; histidine, hydroxide; histidinate, hydroxide). The EPR spectra of the aqua complex reveal that a low-spin state (S = 1/2) and a quantum-mechanically admixed spin state (S = 3/2, 5/2) comprising 12 to 22% S = 3/2 character are thermally accessible at 77 K and room temperature. At pH 10.8 the hydroxo complex has similar crystal field parameters to the aqua complex in both the S = 1/2 and S = 3/2, 5/2 states. At very high pH, the histidinate–hydroxide complex is predominantly low-spin although the S = 3/2, 5/2 state, amounting to less than 20% of the sample at pH 14.3 (77 K), still exhibits significant S = 3/2 character. It is, therefore, demonstrated that a thermal spin-equilibrium between the S = 1/2 and S = 3/2, 5/2 states exists for the three principal pH-dependent forms of AcMP8 above pH 6. This was confirmed by the Mössbauer spectra of lyophilised AcMP8. The effective magnetic moment of monomeric AcMP8 in 23% alcohol–water solution, determined by NMR methods at 25 °C, showed two pH*-dependent transitions above neutral pH*. The first transition (pK_a = 7.83) was assigned to ionisation of iron-bound water (µ^eff = 5.33µ_B) and formation of the hydroxo species (µ^eff = 4.09µ_B). The second transition (pK_a = 10.9) was assigned to ionisation of coordinated histidine and formation of the histidinate–hydroxide complex (µ^eff = 3.16µ_B). The neutral and alkaline forms of AcMP8 are equilibrium mixtures of S = 3/2, 5/2 and S = 1/2 species and the nature of the axial ligand field determines the spin distribution at this temperature. Magnetic susceptibility data collected to 6 K using a SQUID susceptometer revealed that the spin-distribution between the S = 3/2, 5/2 and S = 1/2 states was temperature dependent for both a lyophilised [µ^eff = 2.87µ_B (6 K), µ^eff = 3.83µ_B (297 K)] and a concentrated solution sample of the haempeptide at pH 6 [µ^eff = 2.68µ_B (6 K), µ^eff = 2.81µ_B (182 K)].