Manganese peroxidase (MnP) from Phanerochaete chrysosporium undergoes a pH-dependent conformational change evidenced by changes in the electronic absorption spectrum. This high- to low-spin alkaline transition occurs at ~2 pH units lower in an F190I mutant MnP when compared to the wild-type enzyme. Herein, we provide evidence that these spectral changes are attributable to the formation of a bis(histidyl) heme iron complex in both proteins 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 b558, a protein that contains a bis-His iron complex. Upon reduction with dithionite at high pH, the visible spectra of both the wild-type and F190I MnP exhibit absorption maxima at 429, 529, and 558 nm, resembling the absorption spectrum of ferrous cytochrome b558. RR · spectra of the reduced wild-type and F190I mutant proteins at high pH are also similar to the RR spectrum of ferrous cytochrome b558, further suggesting that the alkaline low-spin species is a bis(histidyl) heme derivative. No shift in the low-frequency RR bands was observed in 75% 18O-labeled water, indicating that the low-spin species is most likely not a hydroxo-heme derivative. Electronic and RR spectra also indicate that addition of Ca2+ to either the ferric or ferrous enzymes at high pH completely restores the high-spin pentacoordinate species. Other divalent metals, such as Mn2+, Mg2+, Zn2+, or Cd2+, do not restore the enzyme under the conditions studied.
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