Crystal Structure of Nitrosomonas europaea Cytochrome c Peroxidase and the Structural Basis for Ligand Switching in Bacterial Di-heme Peroxidases

Abstract

The crystal structure of the fully oxidized di-heme peroxidase from Nitrosomonas europaea has been solved to a resolution of 1.80 A and compared to the closely related enzyme from Pseudomonas aeruginosa. Both enzymes catalyze the peroxide-dependent oxidation of a protein electron donor such as cytochrome c. Electrons enter the enzyme through the high-potential heme followed by electron transfer to the low-potential heme, the site of peroxide activation. Both enzymes form homodimers, each of which folds into two distinct heme domains. Each heme is held in place by thioether bonds between the heme vinyl groups and Cys residues. The high-potential heme in both enzymes has Met and His as axial heme ligands. In the Pseudomonas enzyme, the low-potential heme has two His residues as axial heme ligands [Fulop et al. (1995) Structure 3, 1225-1233]. Since the site of reaction with peroxide is the low-potential heme, then one His ligand must first dissociate. In sharp contrast, the low-potential heme in the Nitrosomonas enzyme already is in the "activated" state with only one His ligand and an open distal axial ligation position available for reaction with peroxide. A comparison between the two enzymes illustrates the range of conformational changes required to activate the Pseudomonas enzyme. This change involves a large motion of a loop containing the dissociable His ligand from the heme pocket to the molecular surface where it forms part of the dimer interface. Since the Nitrosomonas enzyme is in the active state, the structure provides some insights on residues involved in peroxide activation. Most importantly, a Glu residue situated near the peroxide binding site could possibly serve as an acid-base catalytic group required for cleavage of the peroxide O--O bond.

A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea

“…We note the observed pK a values are similar to those observed in a monoheme CcP (24), as well as those found for an active site histidine and bound Fe(III) aquo in studies of myoglobin (25). However, the source of the pK a values are not clear currently, while the superfamily of peroxidases typically contains a conserved Arg and His motif at the peroxide binding site; NeCcP contains an Gln and Glu residue (14). Thus, further experiments will be needed to determine the identity of the specific proton donors to the NeCcP active site.…”

“…However, NeCcP is unlike most other bacterial CcP enzymes, as NeCcP is fully active in the all oxidized, isolated form (13). This property is understood somewhat from consideration of the crystal structure of the enzyme, which possesses an open coordination site at L III (14). Conversely, the structure of the P. aeruginosa CcP (PaCcP) shows that the heme iron of L is coordinately saturated (15) in the fully oxidized and inactive state (Fig.…”

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…Actually, in bacterial enzymes containing more than one domain, such as bacterial cytochrome c peroxidase and nitrite reductase cytochrome cd 1 , it has been observed that reduction of the redox center in one of the domains implies a structural change in the protein [62][63][64][65]. Therefore, similar behavior cannot be ruled out for this enzyme and further experiments are currently in progress in our laboratory to test this hypothesis.…”

Structural and Mutagenesis Studies on the Cytochrome c Peroxidase from Rhodobacter capsulatus Provide New Insights into Structure-Function Relationships of Bacterial Di-heme Peroxidases

“…This indicates that the peroxidatic iron remains bound to a strong field ligand, preventing access of hydrogen peroxide. Located at the N-terminal end of the 108 -118 flexible region is Gln-107, which has been implicated in interactions with diatomic ligands (9). Substitution of this residue with leucine also resulted in an inactive enzyme with a low spin, low potential heme in the mixed valance and in the completely reduced state (Table 2A).…”

“…The high potential heme, with a midpoint potential of ϩ270 mV, therefore, functions as the electron transferring heme, whereas the low potential heme has a midpoint potential between Ϫ190 and Ϫ310 mV (7) and is the peroxidatic center. Structures of the bacterial cytochrome c peroxidases isolated from Pseudomonas aeruginosa (Pa) (8), N. europaea (9), and Pseudomonas nautica (Pn) (10) have already been determined. The structure of the P. aeruginosa enzyme is that of the completely oxidized, inactive enzyme with bound calcium.…”