The quantum mixed-spin heme stale of barley peroxidase: A paradigm for class III peroxidases

Electronic absorption and resonance Raman (RR) spectra of the ferric form o f barley grain peroxidase (BP 1) at various pH values, at both room tempera ture and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride hav e also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relat ive populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl g roups are differently conjugated with the porphyrin. An analysis of the cur rently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as w ell as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influe nces of heme deformations on heme spin state. It is found that moderate sad dling alone is probably not enough to cause the QS state, although some sad dling may be necessary for the QS state.