Formation of a Bis(histidyl) Heme Iron Complex in Manganese Peroxidase at High pH and Restoration of the Native Enzyme Structure by Calcium

Abstract

Manganese peroxidase (MnP) from Phanerochaete chrysosporium undergoes a pH-dependent conformational change evidenced by changes in the electronic absorption spectrum. This high-to lowspin 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 b 558 , 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 b 558 . RR spectra of the reduced wild-type and F190I mutant proteins at 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) heme derivative. No shift in the low-frequency RR bands was observed in 75% 18 O-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 Ca 2+ to either the ferric or ferrous enzymes at high pH completely restores the high-spin pentacoordinate species. Other divalent metals, such as Mn 2+ , Mg 2+ , Zn 2+ , or Cd 2+ , do not restore the enzyme under the conditions studied.Manganese peroxidase (MnP) 1 is an extracellular heme protein produced by virtually all lignin-degrading white-rot fungi (1-3). Sequences have been determined for mnp cDNA (4, 5) and genomic clones (6-8) encoding three MnP isozymes from Phanerochaete chrysosporium, the beststudied lignin-degrading fungus. These sequences and spectroscopic studies of the native and oxidized enzyme intermediates indicate that the heme environment of MnP is similar to that of other plant and fungal peroxidases (4,(9)(10)(11)(12)(13)(14). Kinetic and spectroscopic studies of the purified enzyme indicate a typical peroxidase reaction catalytic cycle:However, MnP is unique in its ability to efficiently oxidize Mn 2+ to Mn 3+ (9,15,16). The released Mn 3+ is stabilized by organic acid chelators, such as oxalate and malonate, which are secreted by the fungus (16, 17). The Mn 3+ -chelator complex is capable of diffusing from the enzyme to oxidize terminal substrates including lignin substructures, phenolic compounds, and pollutants (2, 3 and references therein, 18).The three amino acid residues believed to bind Mn 2+ , D179, E35, and E39, were investigated by site-directed mutagenesis of recombinant MnP homologously expressed in P. chrysosporium (19)(20)(21)(22). These studies, combined with X-ray crystal structure analyses of the proteins (23,24), confirmed that the side-chain carboxylates of D179, E35, and E39 form the only apparent Mn 2+ -binding site in MnP from P. chry...

Disruption of an Active Site Hydrogen Bond Converts Human Heme Oxygenase-1 into a Peroxidase

“…These results support formation of a bis-histidine heme iron complex in the D140H mutant, although in the Fe(II) state the bis-histidine complex is labile, and the engineered histidyl ligand can be displaced by exogenous ligands such as CO and O 2 . The formation of a similar complex was recently reported in the high pH form of manganese peroxidase, where the distal histidine also acts as a labile iron ligand (19). Aside from the D140H mutant, the Fe(II) state of the Asp-140 mutants studied here presents high frequency RR spectra indicative of a 5cHS heme configuration and display in the low frequency region (Fe(II)-His) vibrations within 2 cm Ϫ1 of the 216-cm Ϫ1 value observed for wild-type hHO-1 (data not shown).…”

Regulation of Aldoxime Dehydratase Activity by Redox-dependent Change in the Coordination Structure of the Aldoxime-Heme Complex

“…This reaction intermediate was the ES complex in which PAOx is bound to the ferrous heme. This ES complex showed ␣ and ␤ bands at 555 and 524 nm, respectively, with the Soret peak at 415 nm, which was a typical spectrum of a bis-N coordinate Fe(II) heme (40,41). As shown in Fig.…”

“…Rapid scanning spectroscopy using a stopped-flow system reveals a reaction intermediate that shows the Soret peak at 415 nm. This reaction intermediate is an ES complex where the nitrogen atom of PAOx is bound to the heme (40,41). An important role of the ferrous heme is to tether PAOx in the appropriate orientation toward a catalytic residue(s) in the distal heme pocket.…”

Conformational Mobility in the Active Site of a Heme Peroxidase

“…In line with the above considerations, there are no known examples of a genuine heme peroxidase with bis-histidine ligation, but there are a few examples in the literature of different heme peroxidases, or site-directed variants thereof, in which coordination of the distal histidine residue has been proposed on the basis of spectroscopic studies (3)(4)(5)(6)(7)(8). These examples include: the W51A (7) and D235N (6) variants of cytochrome c peroxidase; thermally inactivated manganese peroxidase (4); and manganese peroxidase at alkaline pH (8).…”

“…These examples include: the W51A (7) and D235N (6) variants of cytochrome c peroxidase; thermally inactivated manganese peroxidase (4); and manganese peroxidase at alkaline pH (8). Removal of the ligands coordinating to the bound K ϩ -site in ascorbate peroxidase also leads to formation of a low-spin species (3).…”