Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

The production of cytochrome c peroxidase (CCP) from Pseudomonas (Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome C-551 (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus (Pa.) denitrificans was proposed to have two different Ca2+ binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca2+. The affinity for Ca2+ in the mixed valence enzyme is so high that Ca2+ returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca2+ for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca2+ in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca2+ does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome C-551) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca2+ binding site of low affinity.

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K3Fe-(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K3Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNIr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe3+ ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site.

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F-peroxo, formed within 25 ms, which then leads to the release of H2O2 and formation of ferric mineral precursors. The stoichiometric relationship between F-peroxo, H2O2, and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H2O2. By rapidly mixing apo M ferritin from frog, Fe2+, and O-2 and allowing the reaction to proceed for 70 ms when F-peroxo has reached its maximum accumulation, followed by spraying the reaction mixture into the H2O2 assay solution, we were able to quantitatively determine the amount of H2O2 produced during the decay of F-peroxo. The correlation between the amount of H2O2 released with the amount of F-peroxo accumulated at 70 ms determined by Mossbauer spectroscopy showed that F-peroxo decays into H2O2 with a stoichiometry of 1 F-peroxo:H2O2. When the decay of F-peroxo was monitored by rapid freeze-quench Mossbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F-peroxo. This observed parallel formation of multiple products (H2O2, diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F-peroxo) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.