Oxygen-18 Kinetic Isotope Effects of Nonheme Iron Enzymes HEPD and MPnS Support Iron(III) Superoxide as the Hydrogen Abstraction Species.

Nonheme iron oxygenases that carry out four-electron oxidations of substrate have been proposed to employ iron(III) superoxide species to initiate this reaction [Paria, S.; Que, L.; Paine, T. K. Angew. Chem. Int. Ed. 2011, 50, 11129]. Here we report experimental evidence in support of this proposal. (18)O KIEs were measured for two recently discovered mononuclear nonheme iron oxygenases: hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS). Competitive (18)O KIEs measured with deuterated substrates are larger than those measured with unlabeled substrates, which indicates that C-H cleavage must occur before an irreversible reductive step at molecular oxygen. A similar observation was previously used to implicate copper(II) superoxide in the H-abstraction reactions catalyzed by dopamine ß-monooxygenase [Tian, G. C.; Klinman, J. P. J. Am. Chem. Soc. 1993, 115, 8891] and peptidylglycine α-hydroxylating monooxygenase [Francisco, W. A.; Blackburn, N. J.; Klinman, J. P. Biochemistry 2003, 42, 1813].

Formation of high-valent iron-oxo species in superoxide reductase: characterization by resonance Raman spectroscopy.

Superoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K48 and I118, led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H2O2. These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O-O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site.

Hydrogen bonding to the cysteine ligand of superoxide reductase: acid-base control of the reaction intermediates.

Superoxide reductase (SOR) is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe(2+) center in a [His4Cys1] square pyramidal pentacoordination, with the axial cysteine ligand proposed to be an essential feature in catalysis. Two NH peptide groups from isoleucine 118 and histidine 119 establish hydrogen bonds involving the sulfur ligand (Desulfoarculus baarsii SOR numbering). To investigate the catalytic role of these hydrogen bonds, the isoleucine 118 residue of the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate, or serine residues. Resonance Raman spectroscopy showed that the mutations specifically induced an increase of the strength of the Fe(3+)-S(Cys) and S-Cß(Cys) bonds as well as a change in conformation of the cysteinyl side chain, which was associated with the alteration of the NH hydrogen bonding involving the sulfur ligand. The effects of the isoleucine mutations on the reactivity of SOR with O2 (•-) were investigated by pulse radiolysis. These studies showed that the mutations induced a specific increase of the pK a of the first reaction intermediate, recently proposed to be an Fe(2+)-O2 (•-) species. These data were supported by density functional theory calculations conducted on three models of the Fe(2+)-O2 (•-) intermediate, with one, two, or no hydrogen bonds involving the sulfur ligand. Our results demonstrated that the hydrogen bonds between the NH (peptide) and the cysteine ligand tightly control the rate of protonation of the Fe(2+)-O2 (•-) reaction intermediate to form an Fe(3+)-OOH species.

Multistep, eight-electron oxidation catalyzed by the cofactorless oxidase, PqqC: identification of chemical intermediates and their dependence on molecular oxygen.

The final step of the biosynthesis of prokaryotic cofactor PQQ is catalyzed by PqqC, a cofactorless oxidase that brings about a ring closure and overall eight-electron oxidation of its substrate. Time-dependent acid quenching and subsequent high-performance liquid chromatography separation and mass spectrometric analyses of reaction mixtures were performed to correlate the structures of intermediates with previously observed UV-visible signatures. The reaction is composed of four stepwise oxidations: three steps use O2 as the two-electron acceptor, and the fourth uses hydrogen peroxide (H2O2). The chemical nature of the intermediates, the stoichiometry of the reaction, and their dependence on the oxygen concentration indicate that the third oxidation uses the product, H2O2, from the preceding step to produce water. The last oxidation step can also be studied separately and is a reaction between O2 and PQQH2 trapped in the active site. This oxidation is approximately 10 times slower than the reoxidation of PQQH2 in solution. From the order of the four oxidation steps and their sensitivity to O2 concentration, we propose a progressive closure of the active site as the enzyme proceeds through its catalytic cycle.

Control of the evolution of iron peroxide intermediate in superoxide reductase from Desulfoarculus baarsii. Involvement of lysine 48 in protonation.

Superoxide reductase is a nonheme iron metalloenzyme that detoxifies superoxide anion radicals O(2)(•-) in some microorganisms. Its catalytic mechanism was previously proposed to involve a single ferric iron (hydro)peroxo intermediate, which is protonated to form the reaction product H(2)O(2). Here, we show by pulse radiolysis that the mutation of the well-conserved lysine 48 into isoleucine in the SOR from Desulfoarculus baarsii dramatically affects its reaction with O(2)(•-). Although the first reaction intermediate and its decay are not affected by the mutation, H(2)O(2) is no longer the reaction product. In addition, in contrast to the wild-type SOR, the lysine mutant catalyzes a two-electron oxidation of an olefin into epoxide in the presence of H(2)O(2), suggesting the formation of iron-oxo intermediate species in this mutant. In agreement with the recent X-ray structures of the peroxide intermediates trapped in a SOR crystal, these data support the involvement of lysine 48 in the specific protonation of the proximal oxygen of the peroxide intermediate to generate H(2)O(2), thus avoiding formation of iron-oxo species, as is observed in cytochrome P450. In addition, we proposed that the first reaction intermediate observed by pulse radiolysis is a ferrous-iron superoxo species, in agreement with TD-DFT calculations of the absorption spectrum of this intermediate. A new reaction scheme for the catalytical mechanism of SOR with O(2)(•-) is presented in which ferrous iron-superoxo and ferric hydroperoxide species are reaction intermediates, and the lysine 48 plays a key role in the control of the evolution of iron peroxide intermediate to form H(2)O(2).

Distribution and properties of the genes encoding the biosynthesis of the bacterial cofactor, pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ) is a small, redox active molecule that serves as a cofactor for several bacterial dehydrogenases, introducing pathways for carbon utilization that confer a growth advantage. Early studies had implicated a ribosomally translated peptide as the substrate for PQQ production. This study presents a sequence- and structure-based analysis of the components of the pqq operon. We find the necessary components for PQQ production are present in 126 prokaryotes, most of which are Gram-negative and a number of which are pathogens. A total of five gene products, PqqA, PqqB, PqqC, PqqD, and PqqE, are identified as being obligatory for PQQ production. Three of the gene products in the pqq operon, PqqB, PqqC, and PqqE, are members of large protein superfamilies. By combining evolutionary conservation patterns with information from three-dimensional structures, we are able to differentiate the gene products involved in PQQ biosynthesis from those with divergent functions. The observed persistence of a conserved gene order within analyzed operons strongly suggests a role for protein-protein interactions in the course of cofactor biosynthesis. These studies propose previously unidentified roles for several of the gene products, as well as identifying possible new targets for antibiotic design and application.

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site.

Superoxide reductase (SOR) is a superoxide detoxification system present in some microorganisms. Its active site consists of an unusual mononuclear iron center with an FeN4S1 coordination which catalyzes the one-electron reduction of superoxide to form hydrogen peroxide. Different classes of SORs have been described depending on the presence of an additional rubredoxin-like, desulforedoxin iron center, whose function has remained unknown until now. In this work, we investigated the mechanism of the reduction of the SOR iron active site using the NADPH:flavodoxin oxidoreductase from Escherichia coli, which was previously shown to efficiently transfer electrons to the Desulfoarculus baarsii SOR. When present, the additional rubredoxin-like iron center could function as an electronic relay between cellular reductases and the iron active site for superoxide reduction. This electron transfer was mainly intermolecular, between the rubredoxin-like iron center of one SOR and the iron active site of another SOR. These data provide the first experimental evidence for a possible role of the rubredoxin-like iron center in the superoxide detoxifying activity of SOR.

Photochemical processes observed during the reaction of superoxide reductase from Desulfoarculus baarsii with superoxide: re-evaluation of the reaction mechanism.

Superoxide reductase SOR is an enzyme involved in superoxide detoxification in some microorganisms. Its active site consists of a non-heme ferrous center in an unusual [Fe(NHis)(4) (SCys)(1)] square pyramidal pentacoordination that efficiently reduces superoxide into hydrogen peroxide. In previous works, the reaction mechanism of the SOR from Desulfoarculus baarsii enzyme, studied by pulse radiolysis, was shown to involve the formation of two reaction intermediates T1 and T2. However, the absorption spectrum of T2 was reported with an unusual sharp band at 625 nm, very different from that reported for other SORs. In this work, we show that the sharp band at 625 nm observed by pulse radiolysis reflects the presence of photochemical processes that occurs at the level of the transient species formed during the reaction of SOR with superoxide. These processes do not change the stoichiometry of the global reaction. These data highlight remarkable photochemical properties for these reaction intermediates, not previously suspected for iron-peroxide species formed in the SOR active site. We have reinvestigated the reaction mechanism of the SOR from D. baarsii by pulse radiolysis in the absence of these photochemical processes. The T1 and T2 intermediates now appear to have absorption spectra similar to those reported for the Archaeoglobus fulgidus SOR enzymes. Although for some enzymes of the family only one transient was reported, on the whole, the reaction mechanisms of the different SORs studied so far seem very similar, which is in agreement with the strong sequence and structure homologies of their active sites.