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.

Nitrosocyanin (NC) is a mononuclear red copper protein isolated from the ammonia oxidizing bacterium Nitrosomonas europaea. Although NC exhibits some sequence homology to classic blue copper proteins, its spectroscopic and electrochemical properties are drastically different. The 1.65 A resolution crystal structure of oxidized NC reveals an unprecedented trimer of single domain cupredoxins. Each copper center is partially covered by an unusual extended beta-hairpin structure from an adjacent monomer. The copper ion is coordinated by His 98, His 103, Cys 95, a single side chain oxygen of Glu 60, and a solvent molecule. In the 2.3 A resolution structure of reduced NC, His 98 shifts away from the copper ion, and the solvent molecule is not observed. The arrangement of these ligands renders the coordination geometry of the NC red copper center distinct from that of blue copper centers. In particular, the red copper center has a higher coordination number and lacks the long Cu-S(Met) and short Cu-S(Cys) bond distances characteristic of blue copper. Moreover, the red copper center is square pyramidal whereas blue copper is typically distorted tetrahedral. Analysis of the NC structure provides insight into possible functions of this new type of biological copper center.

We have determined the 1.8 Å X-ray crystal structure of a monoheme c-type cytochrome, cytochrome P460, from Nitrosomonas europea. The chromophore possesses unusual spectral properties analogous to those of the catalytic heme P460 of hydroxylamine oxidoreductase (HAO), the only known heme in biology to withdraw electrons from an iron-coordinated substrate. The analysis reveals a homodimeric structure and elucidates a new c-type cytochrome fold that is predominantly -sheet. In addition to the two cysteine thioether links to the porphyrin typical of c-type hemes, there is a third proteinaceous link involving a conserved lysine. The covalent bond is between the lysine side-chain nitrogen and the 13′-meso carbon of the heme, which, following cross-link formation, is sp 3 -hybridized, demonstrating the loss of conjugation at this position within the porphyrin. The structure has implications for the analogous tyrosine-heme meso carbon cross-link observed in HAO.

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is an essential electron transfer component in the biological oxidation of ammonia. The protein contains one 5-coordinate heme and three bis-His coordinated hemes in a 3D arrangement common to a newly characterized class of multiheme proteins. The ligand binding, electrochemical properties, and heme-heme interactions are investigated with Mössbauer and X- and Q-band (parallel/perpendicular mode) EPR spectroscopy. The results indicate that the 5-coordinate heme will not bind the common heme ligands, CN(-), F(-), CO, and NO in a wide pH range. Thus, cyt c(554) functions only in electron transfer. Analysis of a series of electrochemically poised and chemically reduced samples allows assignment of reduction potentials for heme 1 through 4 of +47, +47, -147, and -276 mV, respectively. The spectroscopic results indicate that the hemes are weakly exchange-coupled (J approximately -0.5 cm(-)(1)) in two separate pairs and in accordance with the structure: hemes 2/4 (high-spin/low-spin), hemes 1/3 (low-spin/low-spin). There is no evidence of exchange coupling between the pairs. A comparison of the reduction potentials between homologous hemes of cyt c(554) and other members of this new class of multiheme proteins is discussed. Heme 1 has a unique axial N(delta)-His coordination which contributes to a higher potential relative to the homologous hemes of hydroxylamine oxidoreductase (HAO) and the split-Soret cytochrome. Heme 2 is 300 mV more positive than heme 4 of HAO, which is attributed to hydroxide coordination to heme 4 of HAO.