Formation of a bis(histidyl) heme iron complex in manganese peroxidase at high pH and restoration of the native enzyme structure by calcium

Manganese peroxidase (MnP) from Phanerochaete chrysosporium undergoes a pH- dependent conformational change evidenced by changes in the electronic abso rption spectrum. This high- to low-spin alkaline transition occurs at simil ar to 2 pH units lower in an F190I mutant MnP when compared to the wild-typ e enzyme. Herein, we provide evidence that these spectral changes are attri butable to the formation of a bis(histidyl) heme iron complex in both prote ins at high pH. The resonance Raman (RR) spectra of both ferric proteins at high pH are similar, indicating similar heme environments in both proteins , and resemble that of ferric cytochrome b(558), a protein that contains a bis-His iron complex. Upon reduction with dithionite at high pH, the visibl e spectra of both the wild-type and F190I MnP exhibit absorption maxima at 429, 529, and 558 MI, resembling the absorption spectrum of ferrous cytochr ome b(558). RR spectra of the reduced wild-type and F190I mutant proteins a t high pH are also similar to the RR spectrum of ferrous cytochrome b(558), further suggesting that the alkaline low-spin species is a bis(histidyl) h eme derivative. No shift in the low-frequency RR bands was observed in 75% O-18-labeled water, indicating that the low-spin species is most likely not a hydroxo-heme derivative. Electronic and RR spectra also indicate that ad dition of Ca2+ to either the ferric or ferrous enzymes at high pH completel y restores the high-spin pentacoordinate species. Other divalent metals, su ch as Mn2+, Mg2+, Zn2+, or Cd2+, do not restore the enzyme under the condit ions studied.