Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen.

Since the characterization of cytochrome c552 as a multiheme nitrite reductase, research on this enzyme has gained major interest. Today, it is known as pentaheme cytochrome c nitrite reductase (NrfA). Part of the NH4+ produced from NO2- is released as NH3 leading to nitrogen loss, similar to denitrification which generates NO, N2O, and N2. NH4+ can also be used for assimilatory purposes, thus NrfA contributes to nitrogen retention. It catalyses the six-electron reduction of NO2- to NH4+, hosting four His/His ligated c-type hemes for electron transfer and one structurally differentiated active site heme. Catalysis occurs at the distal side of a Fe(III) heme c proximally coordinated by lysine of a unique CXXCK motif (Sulfurospirillum deleyianum, Wolinella succinogenes) or, presumably, by the canonical histidine in Campylobacter jejeuni. Replacement of Lys by His in NrfA of W. succinogenes led to a significant loss of enzyme activity. NrfA forms homodimers as shown by high resolution X-ray crystallography, and there exist at least two distinct electron transfer systems to the enzyme. In γ-proteobacteria (Escherichia coli) NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a pentaheme electron carrier (NrfB), in δ- and ε-proteobacteria (S. deleyianum, W. succinogenes), the NrfA dimer interacts with a tetraheme cytochrome c (NrfH). Both form a membrane-associated respiratory complex on the extracellular side of the cytoplasmic membrane to optimize electron transfer efficiency. This minireview traces important steps in understanding the nature of pentaheme cytochrome c nitrite reductases, and discusses their structural and functional features.

Histidine-Gated Proton-Coupled Electron Transfer to the CuA Site of Nitrous Oxide Reductase.

Copper-containing nitrous oxide reductase (N2OR) is the only known enzyme to catalyze the conversion of the environmentally critical greenhouse gas nitrous oxide (N2O) to dinitrogen (N2) as the final step of bacterial denitrification. Other than its unique tetranuclear active site CuZ, the binuclear electron entry point CuA is also utilized in other enzymes, including cytochrome c oxidase. In the CuA site of Pseudomonas stutzeri N2OR, a histidine ligand was found to undergo a conformational flip upon binding of the substrate N2O between the two copper centers. Here we report on the systematic mutagenesis and spectroscopic and structural characterization of this histidine and surrounding H-bonding residues, based on an established functional expression system for PsN2OR in E. coli. A single hydrogen bond from Ser550 is sufficient to stabilize an unbound conformation of His583, as shown in a Asp576Ala variant, while the additional removal of the hydrogen bond in a Asp576Ala/Ser550Ala double variant compelled His583 to stay in a bound conformation as a ligand to CuA. Systematic mutagenesis of His583 to Ala, Asp, Asn, Glu, Gln, Lys, Phe, Tyr, and Trp showed that although both the CuZ and CuA sites were present in all the variants, only the ones with a protonable side chain, i.e., His, Asp, and Glu, were able to mediate electron transfer at physiological pH. This observation is in line with a proton-coupled electron transfer mechanism at the CuA site of N2OR.

Walking the seven lines: binuclear copper A in cytochrome c oxidase and nitrous oxide reductase.

The enzymes nitrous oxide reductase (N2OR) and cytochrome c oxidase (COX) are constituents of important biological processes. N2OR is the terminal reductase in a respiratory chain converting N2O to N2 in denitrifying bacteria; COX is the terminal oxidase of the aerobic respiratory chain of certain bacteria and eukaryotic organisms transforming O2 to H2O accompanied by proton pumping. Different spectroscopies including magnetic resonance techniques, were applied to show that N2OR has a mixed-valent Cys-bridged [Cu1.5+(CyS)2Cu1.5+] copper site, and that such a binuclear center, called CuA, does also exist in COX. A sequence motif shared between the CuA center of N2OR and the subunit II of COX raises the issue of a putative evolutionary relationship of the two enzymes. The suggestion of a binuclear CuA in COX, with one unpaired electron delocalized between two equivalent Cu nuclei, was difficult to accept originally, even though regarded as a clever solution to many experimental observations. This minireview in honor of Helmut Sigel traces several of the critical steps forward in understanding the nature of CuA in N2OR and COX, and discusses its unique electronic features to some extent including the contributions made by the development of methodology and the discovery of a novel multi-copper enzyme. Left: X-band (9.130 GHz) and C-band (4.530 GHz, 1st harmonic display of experimental spectrum) EPR spectra of bovine heart cytochrome c oxidase, recorded at 20K. Right: Ribbon presentation of the CuA domain in cytochrome c oxidase and nitrous oxide reductase.

N2O binding at a [4Cu:2S] copper-sulphur cluster in nitrous oxide reductase.

Nitrous oxide (N(2)O) is generated by natural and anthropogenic processes and has a critical role in environmental chemistry. It has an ozone-depleting potential similar to that of hydrochlorofluorocarbons as well as a global warming potential exceeding that of CO(2) 300-fold. In bacterial denitrification, N(2)O is reduced to N(2) by the copper-dependent nitrous oxide reductase (N(2)OR). This enzyme carries the mixed-valent Cu(A) centre and the unique, tetranuclear Cu(Z) site. Previous structural data were obtained with enzyme isolated in the presence of air that is catalytically inactive without prior reduction. Its Cu(Z) site was described as a [4Cu:S] centre, and the substrate-binding mode and reduction mechanism remained elusive. Here we report the structure of purple N(2)OR from Pseudomonas stutzeri, handled under the exclusion of dioxygen, and locate the substrate in N(2)O-pressurized crystals. The active Cu(Z) cluster contains two sulphur atoms, yielding a [4Cu:2S] stoichiometry; and N(2)O bound side-on at Cu(Z), in close proximity to Cu(A). With the substrate located between the two clusters, electrons are transferred directly from Cu(A) to N(2)O, which is activated by side-on binding in a specific binding pocket on the face of the [4Cu:2S] centre. These results reconcile a multitude of available biochemical data on N(2)OR that could not be explained by earlier structures, and outline a mechanistic pathway in which both metal centres and the intervening protein act in concert to achieve catalysis. This structure represents the first direct observation, to our knowledge, of N(2)O bound to its reductase, and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates. The principle of using distinct clusters for substrate activation and for reduction may be relevant for similar systems, in particular nitrogen-fixing nitrogenase.

Acetylene hydratase: a non-redox enzyme with tungsten and iron-sulfur centers at the active site.

In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe-4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26 Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C2H2. To access the W-Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C2H2 binds directly to W. The architecture of the catalytic cavity, the specificity for C2H2 and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C2H2 are needed to present a conclusive answer.

The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.

In vivo speciation studies and antioxidant properties of bromine in Laminaria digitata reinforce the significance of iodine accumulation for kelps.

The metabolism of bromine in marine brown algae remains poorly understood. This contrasts with the recent finding that the accumulation of iodide in the brown alga Laminaria serves the provision of an inorganic antioxidant - the first case documented from a living system. The aim of this study was to use an interdisciplinary array of techniques to study the chemical speciation, transformation, and function of bromine in Laminaria and to investigate the link between bromine and iodine metabolism, in particular in the antioxidant context. First, bromine and iodine levels in different Laminaria tissues were compared by inductively coupled plasma MS. Using in vivo X-ray absorption spectroscopy, it was found that, similarly to iodine, bromine is predominantly present in this alga in the form of bromide, albeit at lower concentrations, and that it shows similar behaviour upon oxidative stress. However, from a thermodynamic and kinetic standpoint, supported by in vitro and reconstituted in vivo assays, bromide is less suitable than iodide as an antioxidant against most reactive oxygen species except superoxide, possibly explaining why kelps prefer to accumulate iodide. This constitutes the first-ever study exploring the potential antioxidant function of bromide in a living system and other potential physiological roles. Given the tissue-specific differences observed in the content and speciation of bromine, it is concluded that the bromide uptake mechanism is different from the vanadium iodoperoxidase-mediated uptake of iodide in L. digitata and that its function is likely to be complementary to the iodide antioxidant system for detoxifying superoxide.

Toxicity and deficiency of copper in Elsholtzia splendens affect photosynthesis biophysics, pigments and metal accumulation.

Elsholtzia splendens is a copper-tolerant plant species growing on copper deposits in China. Spatially and spectrally resolved kinetics of in vivo absorbance and chlorophyll fluorescence in mesophyll of E. splendens were used to investigate the copper-induced stress from deficiency and toxicity as well as the acclimation to excess copper stress. The plants were cultivated in nutrient solutions containing either Fe(III)-EDTA or Fe(III)-EDDHA. Copper toxicity affected light-acclimated electron flow much stronger than nonphotochemical quenching (NPQ) or dark-acclimated photochemical efficiency of PSIIRC (Fv/Fm). It also changed spectrally resolved Chl fluorescence kinetics, in particular by strengthening the short-wavelength (<700 nm) part of NPQ altering light harvesting complex II (LHCII) aggregation. Copper toxicity reduced iron accumulation, decreased Chls and carotenoids in leaves. During acclimation to copper toxicity, leaf copper decreased but leaf iron increased, with photosynthetic activity and pigments recovering to normal levels. Copper tolerance in E. splendens was inducible; acclimation seems be related to homeostasis of copper and iron in E. splendens. Copper deficiency appeared at 10 mg copper per kg leaf DW, leading to reduced growth and decreased photosynthetic parameters (F0, Fv/Fm, ΦPSII). The importance of these results for evaluating responses of phytoremediation plants to stress in their environment is discussed.

Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens.

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P(1B)-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10µM), and all three metal ions activated the ATPase at a concentration of 0.3µM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E(A)=38kJ⋅mol(-1)) over the whole concentration range while it increased from 17 to 42kJ⋅mol(-1) with rising copper concentration and decreased from 39 to 23kJ⋅mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.

The catalytic redox activity of prion protein-Cu(II) is controlled by metal exchange with the Zn(II) -thiolate clusters of Zn(7) metallothionein-3.

Silencing prion: Copper-catalyzed transformations of prion protein (PrP) lead to the production of reactive oxygen species (ROS), PrP oxidation, and cleavage and aggregation in transmissible spongiphorm encephalopathies. Zn(7) MT-3 efficiently targets Cu(II) bound in different coordination modes to PrP-Cu(II) . By an unusual redox-dependent metal-swap reaction, MT-3 modulates the catalytic redox properties of PrP-Cu(II) .

Nature's way of handling a greenhouse gas: the copper-sulfur cluster of purple nitrous oxide reductase.

The tetranuclear Cu(Z) cluster is the unique active site of nitrous oxide reductase, the enzyme that catalyzes the reduction of nitrous oxide to dinitrogen as the final reaction in bacterial denitrification. Three-dimensional structures of orthologs of the enzyme from a variety of different bacterial species were essential steps in the elucidation of the properties of this center. However, while structural data first revealed and later confirmed the presence of four copper ions in spectroscopically distinct forms of Cu(Z), the exact structure and stoichiometry of the cluster showed significant variations. A ligand bridging ions Cu(Z1) and Cu(Z2) was initially assigned as a water or hydroxo species in the structures from Pseudomonas nautica (now Marinobacter hydrocarbonoclasticus) and Paracoccus denitrificans. This ligand was absent in a structure from 'Achromobacter cycloclastes', and could be reconstituted by iodide that acted as an inhibitor of catalysis. A recent structure of anoxically isolated nitrous oxide reductase from Pseudomonas stutzeri revealed the bridging ligand to be sulfide, S2-, and showed an unprecedented side-on mode of nitrous oxide binding to this form of Cu(Z).

Exploring the active site of the tungsten, iron-sulfur enzyme acetylene hydratase.

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H-C≡C-H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme.

Cyclohexane-1,2-dione hydrolase from denitrifying Azoarcus sp. strain 22Lin, a novel member of the thiamine diphosphate enzyme family.

Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a C-C bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a C-C bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD(+)-dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ∼59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (∼105 kDa; gel filtration). As isolated, CDH contains 0.8 ± 0.05 ThDP, 1.0 ± 0.02 Mg(2+), and 1.0 ± 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na(+)-dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3- and 1,4-diones did not react with CDH, nor did the cis- and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold for FAD binding could be localized at the C-terminal end and central region of CDH, respectively. A first mechanism for the ring cleavage of CDO is presented, and it is suggested that the FAD cofactor in CDH is an evolutionary relict.

Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry.

Brown algae of the Laminariales (kelps) are the strongest accumulators of iodine among living organisms. They represent a major pump in the global biogeochemical cycle of iodine and, in particular, the major source of iodocarbons in the coastal atmosphere. Nevertheless, the chemical state and biological significance of accumulated iodine have remained unknown to this date. Using x-ray absorption spectroscopy, we show that the accumulated form is iodide, which readily scavenges a variety of reactive oxygen species (ROS). We propose here that its biological role is that of an inorganic antioxidant, the first to be described in a living system. Upon oxidative stress, iodide is effluxed. On the thallus surface and in the apoplast, iodide detoxifies both aqueous oxidants and ozone, the latter resulting in the release of high levels of molecular iodine and the consequent formation of hygroscopic iodine oxides leading to particles, which are precursors to cloud condensation nuclei. In a complementary set of experiments using a heterologous system, iodide was found to effectively scavenge ROS in human blood cells.

A [3Cu:2S] cluster provides insight into the assembly and function of the CuZ site of nitrous oxide reductase.

Nitrous oxide reductase (N2OR) is the only known enzyme reducing environmentally critical nitrous oxide (N2O) to dinitrogen (N2) as the final step of bacterial denitrification. The assembly process of its unique catalytic [4Cu:2S] cluster CuZ remains scarcely understood. Here we report on a mutagenesis study of all seven histidine ligands coordinating this copper center, followed by spectroscopic and structural characterization and based on an established, functional expression system for Pseudomonas stutzeri N2OR in Escherichia coli. While no copper ion was found in the CuZ binding site of variants H129A, H130A, H178A, H326A, H433A and H494A, the H382A variant carried a catalytically inactive [3Cu:2S] center, in which one sulfur ligand, SZ2, had relocated to form a weak hydrogen bond to the sidechain of the nearby lysine residue K454. This link provides sufficient stability to avoid the loss of the sulfide anion. The UV-vis spectra of this cluster are strikingly similar to those of the active enzyme, implying that the flexibility of SZ2 may have been observed before, but not recognized. The sulfide shift changes the metal coordination in CuZ and is thus of high mechanistic interest.

Reaction cycle of the dissimilatory sulfite reductase from Archaeoglobus fulgidus.

A vital process in the biogeochemical sulfur cycle is the dissimilatory sulfate reduction pathway in which sulfate (SO4⁻²) is converted to hydrogen sulfide (H2S). Dissimilatory sulfite reductase (dSir), its key enzyme, hosts a unique siroheme-[4Fe-4S] cofactor and catalyzes the six-electron reduction of sulfite (SO3²â») to H2S. To explore this reaction, we determined the X-ray structures of dSir from the archaeon Archaeoglobus fulgidus in complex with sulfite, sulfide (S²â») carbon monoxide (CO), cyanide (CN⁻), nitrite (NO2⁻), nitrate (NO3⁻), and phosphate (PO4³â»). Activity measurements indicated that dSir of A. fulgidus reduces, besides sulfite and nitrite, thiosulfate (S2O3²â») and trithionate (S3O6²â») and produces the latter two compounds besides sulfide. On this basis, a three-step mechanism was proposed, each step consisting of a two-electron transfer, a two-proton uptake, and a dehydration event. In comparison, the related active site structures of the assimilatory sulfite reductase (aSir)- and dSir-SO3²â»complexes reveal different conformations of Argα170 and Lysα211 both interacting with the sulfite oxygens (its sulfur atom coordinates the siroheme iron), a sulfite rotation of ~60° relative to each other, and different access of solvent molecules to the sulfite oxygens from the active site cleft. Therefore, solely in dSir a further sulfite molecule can be placed in van der Waals contact with the siroheme-ligated sulfite or sulfur-oxygen intermediates necessary for forming thiosulfate and trithionate. Although reported for dSir from several sulfate-reducing bacteria, the in vivo relevance of their formation is questionable.

Spectroscopic properties of rubber oxygenase RoxA from Xanthomonas sp., a new type of dihaem dioxygenase.

Natural rubber [poly-(cis-1,4-isoprene)] is cleaved to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA has two c-type haem centres that show two distinct alpha-bands at 549 and 553 nm in the dithionite-reduced state. A well-resolved midpoint potential (E(0)') of -65 mV was determined for one haem by spectrophotometric titrations in the absence of dioxygen with dithionite and ferricyanide as reductant and oxidant, respectively. The midpoint potential of the second haem was not resolvable (E(0)' about -130 to -160 mV). One of the two haems was reduced by NADH (549 nm alpha-band), similar to bacterial dihaem peroxidases. Evidence for an electron transfer between the two haems was provided by slow reduction of the second haem (553 nm alpha-band) upon incubation of the partially reduced enzyme at room temperature. Addition of imidazole or related compounds to RoxA led to UV/vis spectral features similar to those observed for partially reduced RoxA. Notably, reduction of RoxA with dithionite or NADH, or binding of compounds such as imidazole, resulted in a reversible inactivation of the enzyme, unlike dihaem peroxidases. In line with this result, RoxA did not show any peroxidase activity. EPR spectra of RoxA as isolated showed two low-spin Fe(III) haem centres, with apparent g-values of 3.39, 3.09, 2.23, 1.92 and 1.50. A weak signal in the g=6 region resulting from a high-spin Fe(III) haem was also observed with a preparation-dependent intensity that disappeared in the presence of imidazole. Attempts to provide spectroscopic evidence for binding of the natural substrate (polyisoprene latex) to RoxA failed. However, experimental data are presented that RoxA is able to subtract redox equivalents from its substrate or from model compounds. In conclusion, RoxA is a novel type of dihaem dioxygenase with features clearly different from classical cytochrome c peroxidases.

Crystallization of purple nitrous oxide reductase from Pseudomonas stutzeri.

Nitrous oxide reductase (N(2)OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N(2)OR crystallized in either space group P1 or P6(5). Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130 kDa dimers in the asymmetric unit, while crystals belonging to space group P6(5) contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2 Å.