HAEMPEPTIDE MODELS FOR HEMOPROTEINS - PART 3 N-ACETYLMICROPEROXIDASE-8 - EPR, MOSSBAUER AND MAGNETIC-SUSCEPTIBILITY STUDIES ON AN IRON(III)PORPHYRIN IN THERMAL-EQUILIBRIUM BETWEEN S=3 2,5/2 AND S=1/2 STATES/

The N-terminus acetylated haemoctapeptide from cytochrome c, acetylmic roperoxidase-8 (AcMP8 OH,) displays complex magnetic behaviour, contin gent on its pH-dependent axial ligand combinations (histidine, H2O; hi stidine, hydroxide; histidinate, hydroxide). The EPR spectra of the aq ua complex reveal that a low-spin state (S = 1/2) and a quantum-mechan ically admired 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 altho ugh the S = 3/2, 5/2 state, amounting to less than 20% of the sample a t 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-depen dent forms of AcMP8 above pH 6. This was confirmed by the Mossbauer sp ectra of lyophilised AcMP8. The effective magnetic moment of monomeric AcMP8 in 23% alcohol-water solution, determined by NMR methods at 25 degrees C, showed two pH-dependent transitions above neutral pH*. The first transition (pK(a) = 7.83) was assigned to ionisation of iron-bo und water(mu(eff) = 5.33 mu(B)) and formation of the hydroxo species ( mu(eff) = 4.09 mu(B)). The second transition (pK(a) = 10.9) was assign ed to ionisation of coordinated histidine and formation of the histidi nate-hydroxide complex (mu(eff) = 3.16 mu(B)). The neutral and alkalin e 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 d istribution at this temperature. Magnetic susceptibility data collecte d to 6 K using a SQUID susceptometer revealed that the spin-distributi on between the S = 3/2, 5/2 and S = 1/2 states was temperature depende nt for both a lyophilised [mu(eff) = 2.87 mu(B), (6 K), mu(eff) = 3.83 mu(B), (297 K)] and a concentrated solution sample of the haempeptide at pH 6 [mu(eff) = 2.68 mu(B), (6 K), mu(eff) = 2.81 mu(B), (182 K)].