Site Of Nitric Oxide Reductase Research Articles

Cu4S Cluster in "0-Hole" and "1-Hole" States: Geometric and Electronic Structure Variations for the Active CuZ* Site of N2O Reductase.

The active site of nitrous oxide reductase (N2OR), a key enzyme in denitrification, features a unique μ4-sulfido-bridged tetranuclear Cu cluster (the so-called CuZ or CuZ* site). Details of the catalytic mechanism have remained under debate and, to date, synthetic model complexes of the CuZ*/CuZ sites are extremely rare due to the difficulty in building the unique {Cu4(μ4-S)} core structure. Herein, we report the synthesis and characterization of [Cu4(μ4-S)]n+ (n = 2, 2; n = 3, 3) clusters, supported by a macrocyclic {py2NHC4} ligand (py = pyridine, NHC = N-heterocyclic carbene), in both their 0-hole (2) and 1-hole (3) states, thus mimicking the two active states of the CuZ* site during enzymatic N2O reduction. Structural and electronic properties of these {Cu4(μ4-S)} clusters are elucidated by employing multiple methods, including X-ray diffraction (XRD), nuclear magnetic resonance (NMR), UV/vis, electron paramagnetic resonance (EPR), Cu/S K-edge X-ray emission spectroscopy (XES), and Cu K-edge X-ray absorption spectroscopy (XAS) in combination with time-dependent density functional theory (TD-DFT) calculations. A significant geometry change of the {Cu4(μ4-S)} core occurs upon oxidation from 2 (τ4(S) = 0.46, seesaw) to 3 (τ4(S) = 0.03, square planar), which has not been observed so far for the biological CuZ(*) site and is unprecedented for known model complexes. The single electron of the 1-hole species 3 is predominantly delocalized over two opposite Cu ions via the central S atom, mediated by a π/π superexchange pathway. Cu K-edge XAS and Cu/S K-edge XES corroborate a mixed Cu/S-based oxidation event in which the lowest unoccupied molecular orbital (LUMO) has a significant S-character. Furthermore, preliminary reactivity studies evidence a nucleophilic character of the central μ4-S in the fully reduced 0-hole state.

Dimeric Copper and Lithium Thiolates: Comparison of Copper Thiolates with Their Lithium Congeners.

The direct reactions of the large terphenyl thiols HSAriPr4 (AriPr4= -C6H3-2,6-(C6H3-2,6-iPr2)2) and HSAriPr6 (AriPr6= -C6H3-2,6-(C6H2-2,4,6-iPr3)2) with stoichiometric amounts of mesitylcopper(I) in THF at ca. 80 °C afforded the first well-characterized dimeric copper thiolato species {CuSAriPr4}2 (1) and {CuSAriPr6}2 (2) with elimination of mesitylene. The complexes 1 and 2 were characterized by NMR and electronic spectroscopy as well as by X-ray crystallography. They have dimeric Cu2S2 core structures in which the two copper atoms are bridged by the sulfurs from the thiolato ligands and feature short Cu--Cu distances near 2.4 Å as well as a weak copper-flanking aryl ring interaction from a terphenyl substituent. The structures of the planar Cu2S2 cores bear a resemblance to the CuA site in nitrous oxide reductase in which two cysteines also bridge two copper atoms. The related dimeric Li2S2 structural motif was also observed in the lithium congeners {LiSAriPr4}2 (3) and {LiSAriPr6}2 (4) which were synthesized directly from the thiols and n-BuLi in hexanes. However, despite the very similar effective ionic radii of the Li+ (0.59 Å) and Cu+ (0.60 Å) ions, the Li--Li structures display very much longer (by more than ca. 0.5 Å) separations than the corresponding Cu--Cu distances in 1 and 2, which may be due to weaker dispersion interactions.

Electrocatalytic Reduction of Nitrogen Oxides By a Crystalline Carbon Nitride

Of the trillions of tons of Haber-Bosch fertilizers generated annually for global consumption, only 17% contribute to human feedstock proteins. A large fraction of Haber-Bosch nitrogen is lost as hazardous oxidized waste (N y O x ). Water-soluble NO3 – and NO2 – anions wash into aquatic ecosystems, where they feed dangerous algal blooms. The waste volatile N2O comprised nearly 7% of emissions in 2018, and has 300 times the global warming impact of CO2. Nature mediates N y O x redox through metalloheme chains aligned in enzymatic active sites. The poly(triazine imide) LiCl (PTI-LiCl) electrocatalyst mimics these heme chains through highly crystalline, layered carbon nitride nanosheets. PTI has advantages of both heterogeneous and homogeneous electrocatalysts: (i) PTI sequesters substrates as intercalants, (ii) PTI has high surface area with a known structure, and does not lose crystallinity upon introduction of abundant transition metals, (iii) PTI has an extended π system shuttles electrons to ordered active sites. PTI is active as a solid state electrocatalyst toward NO2 – reduction, with 31% Faradaic efficiency toward complete reduction to NH3. Approximately 50% of intrasheet Li+ can be exchanged with Cu2+ with no change to native crystallinity, suggesting an electrostatic interaction between PTI and Cu2+ single atoms. Remarkably, introducing Cu2+ improves the onset potential for electrocatalytic NO2 – reduction by nearly 0.5 V, and catalytic current increases by an order of magnitude. Since copper is also found at the active site in nitrous oxide reductase, CuPTI was investigated for electrocatalytic reduction of N2O. Preliminary results suggest CuPTI can electrochemically bind and reduce N2O. Figure 1

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.

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.

Impact of Electronic and Steric Changes of Ligands on the Assembly, Stability, and Redox Activity of Cu4(μ4-S) Model Compounds of the CuZ Active Site of Nitrous Oxide Reductase (N2OR).

Model compounds have been widely utilized in understanding the structure and function of the unusual Cu4(μ4-S) active site (CuZ) of nitrous oxide reductase (N2OR). However, only a limited number of model compounds that mimic both structural and functional features of CuZ are available, limiting insights about CuZ that can be gained from model studies. Our aim has been to construct Cu4(μ4-S) clusters with tailored redox activity and chemical reactivity via modulating the ligand environment. Our synthetic approach uses dicopper(I) precursor complexes (Cu2L2) that assemble into a Cu4(μ4-S)L4 cluster with the addition of an appropriate sulfur source. Here, we summarize the features of the ligands L that stabilize precursor and Cu4(μ4-S) clusters, along with the alternative products that form with inappropriate ligands. The precursors are more likely to rearrange to Cu4(μ4-S) clusters when the Cu(I) ions are supported by bidentate ligands with 3-atom bridges, but steric and electronic features of the ligand also play crucial roles. Neutral phosphine donors have been found to stabilize Cu4(μ4-S) clusters in the 4Cu(I) oxidation state, while neutral nitrogen donors could not stabilize Cu4(μ4-S) clusters. Anionic formamidinate ligands have been found to stabilize Cu4(μ4-S) clusters in the 2Cu(I):2Cu(II) and 3Cu(I):1Cu(II) states, with both the formation of the dicopper(I) precursors and subsequent assembly of clusters being governed by the steric factor at the ortho positions of the N-aryl substituents. Phosphaamidinates, which combine a neutral phosphine donor and an anionic nitrogen donor in the same ligand, form multinuclear Cu(I) clusters unless the negative charge is valence-trapped on nitrogen, in which case the resulting dicopper precursor is unable to rearrange to a multinuclear cluster. Taken together, the results presented in this study provide design criteria for successful assembly of synthetic model clusters for the CuZ active site of N2OR, which should enable future insights into the chemical behavior of CuZ.

N2O Reductase Activity of a [Cu4S] Cluster in the 4CuI Redox State Modulated by Hydrogen Bond Donors and Proton Relays in the Secondary Coordination Sphere

AbstractThe model complex [Cu4(μ4‐S)(dppa)4]2+ (1, dppa=μ2‐(Ph2P)2NH) has N2O reductase activity in methanol solvent, mediating 2 H+/2 e− reduction of N2O to N2+H2O in the presence of an exogenous electron donor (CoCp2). A stoichiometric product with two deprotonated dppa ligands was characterized, indicating a key role of second‐sphere N−H residues as proton donors during N2O reduction. The activity of 1 towards N2O was suppressed in solvents that are unable to provide hydrogen bonding to the second‐sphere N−H groups. Structural and computational data indicate that second‐sphere hydrogen bonding induces structural distortion of the [Cu4S] active site, accessing a strained geometry with enhanced reactivity due to localization of electron density along a dicopper edge site. The behavior of 1 mimics aspects of the CuZ catalytic site of nitrous oxide reductase: activity in the 4CuI:1S redox state, use of a second‐sphere proton donor, and reactivity dependence on both primary and secondary sphere effects.

N2 O Reductase Activity of a [Cu4 S] Cluster in the 4CuI Redox State Modulated by Hydrogen Bond Donors and Proton Relays in the Secondary Coordination Sphere.

The model complex [Cu4 (μ4 -S)(dppa)4 ]2+ (1, dppa=μ2 -(Ph2 P)2 NH) has N2 O reductase activity in methanol solvent, mediating 2 H+ /2 e- reduction of N2 O to N2 +H2 O in the presence of an exogenous electron donor (CoCp2 ). A stoichiometric product with two deprotonated dppa ligands was characterized, indicating a key role of second-sphere N-H residues as proton donors during N2 O reduction. The activity of 1 towards N2 O was suppressed in solvents that are unable to provide hydrogen bonding to the second-sphere N-H groups. Structural and computational data indicate that second-sphere hydrogen bonding induces structural distortion of the [Cu4 S] active site, accessing a strained geometry with enhanced reactivity due to localization of electron density along a dicopper edge site. The behavior of 1 mimics aspects of the CuZ catalytic site of nitrous oxide reductase: activity in the 4CuI :1S redox state, use of a second-sphere proton donor, and reactivity dependence on both primary and secondary sphere effects.

Oxidation of a [Cu2S] complex by N2O and CO2: insights into a role of tetranuclearity in the CuZ site of nitrous oxide reductase.

Oxidation of a [Cu2(μ-S)] complex by N2O or CO2 generated a [Cu2(μ-SO4)] product. In the presence of a sulfur trap, a [Cu2(μ-O)] species also formed from N2O. A [Cu2(μ-CS3)] species derived from CS2 modeled initial reaction intermediates. These observations indicate that one role of tetranuclearity in the CuZ catalytic site of nitrous oxide reductase is to protect the crucial S2- ligand from oxidation.

Spectroscopic Definition of the CuZ° Intermediate in Turnover of Nitrous Oxide Reductase and Molecular Insight into the Catalytic Mechanism.

Spectroscopic methods and density functional theory (DFT) calculations are used to determine the geometric and electronic structure of CuZ°, an intermediate form of the Cu4S active site of nitrous oxide reductase (N2OR) that is observed in single turnover of fully reduced N2OR with N2O. Electron paramagnetic resonance (EPR), absorption, and magnetic circular dichroism (MCD) spectroscopies show that CuZ° is a 1-hole (i.e., 3CuICuII) state with spin density delocalized evenly over CuI and CuIV. Resonance Raman spectroscopy shows two Cu-S vibrations at 425 and 413 cm-1, the latter with a -3 cm-1 O18 solvent isotope shift. DFT calculations correlated to these spectral features show that CuZ° has a terminal hydroxide ligand coordinated to CuIV, stabilized by a hydrogen bond to a nearby lysine residue. CuZ° can be reduced via electron transfer from CuA using a physiologically relevant reductant. We obtain a lower limit on the rate of this intramolecular electron transfer (IET) that is >104 faster than the unobserved IET in the resting state, showing that CuZ° is the catalytically relevant oxidized form of N2OR. Terminal hydroxide coordination to CuIV in the CuZ° intermediate yields insight into the nature of N2O binding and reduction, specifying a molecular mechanism in which N2O coordinates in a μ-1,3 fashion to the fully reduced state, with hydrogen bonding from Lys397, and two electrons are transferred from the fully reduced μ4S2- bridged tetranuclear copper cluster to N2O via a single Cu atom to accomplish N-O bond cleavage.

Structure and properties of the catalytic site of nitric oxide reductase at ambient temperature

Nitric oxide reductase (Nor) is the third of the four enzymes of bacterial denitrification responsible for the catalytic formation of laughing gas (N2O). Here we report the detection of the hyponitrite (HO–N=N–O−) species (νN–N=1332cm−1) in the heme b3 Fe–FeB dinuclear center of Nor from Paracoccus denitrificans. We have also applied density functional theory (DFT) to characterize the bimetallic-bridging hyponitrite species in the reduction of NO to N2O by Nor and compare the present results with those recently reported for the N–N bond formation in the ba3 and caa3 oxidoreductases from Thermus thermophilus.

Assembly, structure, and reactivity of Cu₄S and Cu₃S models for the nitrous oxide reductase active site, Cu(Z)*.

Bridging diphosphine ligands were used to facilitate the assembly of copper clusters with single sulfur atom bridges that model the structure of the Cu(Z)* active site of nitrous oxide reductase. Using bis(diphenylphosphino)amine (dppa), a [Cu(I)4(μ4-S)] cluster with N-H hydrogen bond donors in the secondary coordination sphere was assembled. Solvent and anion guests were found docking to the N-H sites in the solid state and in the solution phase, highlighting a kinetically viable pathway for substrate introduction to the inorganic core. Using bis(dicyclohexylphosphino)methane (dcpm), a [Cu(I)3(μ3-S)] cluster was assembled preferentially. Both complexes exhibited reversible oxidation events in their cyclic voltammograms, making them functionally relevant to the Cu(Z)* active site that is capable of catalyzing a multielectron redox transformation, unlike the previously known [Cu(I)4(μ4-S)] complex from Yam and co-workers supported by bis(diphenylphosphino)methane (dppm). The dppa-supported [Cu(I)4(μ4-S)] cluster reacted with N3(-), a linear triatomic substrate isoelectronic to N2O, in preference to NO2(-), a bent triatomic. This [Cu(I)4(μ4-S)] cluster also bound I(-), a known inhibitor of Cu(Z)*. Consistent with previous observations for nitrous oxide reductase, the tetracopper model complex bound the I(-) inhibitor much more strongly and rapidly than the substrate isoelectronic to N2O, producing unreactive μ3-iodide clusters including a [Cu3(μ3-S)(μ3-I)] complex related to the [Cu4(μ4-S)(μ2-I)] form of the inhibited enzyme.

No laughing matter: the unmaking of the greenhouse gas dinitrogen monoxide by nitrous oxide reductase.

The gas nitrous oxide (N₂O) is generated in a variety of abiotic, biotic, and anthropogenic processes and it has recently been under scrutiny for its role as a greenhouse gas. A single enzyme, nitrous oxide reductase, is known to reduce N₂O to uncritical N₂, in a two-electron reduction process that is catalyzed at two unusual metal centers containing copper. Nitrous oxide reductase is a bacterial metalloprotein from the metabolic pathway of denitrification, and it forms a 130 kDa homodimer in which the two metal sites CuA and CuZ from opposing monomers are brought into close contact to form the active site of the enzyme. CuA is a binuclear, valence-delocalized cluster that accepts and transfers a single electron. The CuA site of nitrous oxide reductase is highly similar to that of respiratory heme-copper oxidases, but in the denitrification enzyme the site additionally undergoes a conformational change on a ligand that is suggested to function as a gate for electron transfer from an external donor protein. CuZ, the tetranuclear active center of nitrous oxide reductase, is isolated under mild and anoxic conditions as a unique [4Cu:2S] cluster. It is easily desulfurylated to yield a [4Cu:S] state termed CuZ (*) that is functionally distinct. The CuZ form of the cluster is catalytically active, while CuZ (*) is inactive as isolated in the [3Cu(1+):1Cu(2+)] state. However, only CuZ (*) can be reduced to an all-cuprous state by sodium dithionite, yielding a form that shows higher activities than CuZ. As the possibility of a similar reductive activation in the periplasm is unconfirmed, the mechanism and the actual functional state of the enzyme remain under debate. Using enzyme from anoxic preparations with CuZ in the [4Cu:2S] state, N2O was shown to bind between the CuA and CuZ sites, suggesting direct electron transfer from CuA to the substrate after its activation by CuZ.

Unexpected weak magnetic exchange coupling between haem and non-haem iron in the catalytic site of nitric oxide reductase (NorBC) from Paracoccus denitrificans

Bacterial NOR (nitric oxide reductase) is a major source of the powerful greenhouse gas N2O. NorBC from Paracoccus denitrificans is a heterodimeric multi-haem transmembrane complex. The active site, in NorB, comprises high-spin haem b3 in close proximity with non-haem iron, FeB. In oxidized NorBC, the active site is EPR-silent owing to exchange coupling between FeIII haem b3 and FeBIII (both S=5/2). On the basis of resonance Raman studies [Moënne-Loccoz, Richter, Huang, Wasser, Ghiladi, Karlin and de Vries (2000) J. Am. Chem. Soc. 122, 9344-9345], it has been assumed that the coupling is mediated by an oxo-bridge and subsequent studies have been interpreted on the basis of this model. In the present study we report a VFVT (variable-field variable-temperature) MCD (magnetic circular dichroism) study that determines an isotropic value of J=-1.7 cm-1 for the coupling. This is two orders of magnitude smaller than that encountered for oxo-bridged diferric systems, thus ruling out this configuration. Instead, it is proposed that weak coupling is mediated by a conserved glutamate residue.

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).

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.

Rational design of a structural and functional nitric oxide reductase

SummaryProtein design provides an ultimate test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. While progress has been made in designing proteins that mimic native proteins structurally1–3, it is more difficult to design functional proteins4–8. In comparison to recent successes in designing non-metalloproteins4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes5,8,11–20, since protein metal binding sites are much more varied than non-metal containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal binding site properties in silico, as many of the parameters for metal binding sites, such as force fields are ill-defined. Therefore, the successful design of a structural and functional metalloprotein will greatly advance the field of protein design and our understanding of enzymes. Here, we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a heme/non-heme FeB center that is remarkably similar to that in the crystal structure. This designed protein also exhibits NOR activity. This is the first designed protein that models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

One heme, diverse functions: using biosynthetic myoglobin models to gain insights into heme-copper oxidases and nitric oxide reductases.

The heme-copper oxidase superfamily: Heme-copper oxidase (HCO) and nitric oxide reductase (NOR) Heme-copper oxidases (HCOs) are a superfamily of terminal oxidases present in the respiratory chains of both bacteria and eukaryotic mitochondria.5 HCOs utilize O2 as a terminal electron acceptor, catalyzing the four electron reduction of O2 to H2O (O2 + 4e− + 8H+in → 2H2O + 4H+out), while generating a proton gradient that is used to drive the production of ATP. Nitric oxide reductase (NOR) is a metalloenzyme in the denitrification pathway responsible for the two electron reduction of NO to N2O (2NO + 2H+ + 2e− → N2O + H2O), a reaction that is nonelectrogenic (i.e., no charge translocation across the membrane).6,7 Denitrifying NO reductases are also members of the heme-copper oxidase superfamily. However, despite the diversity of their native functions, NOR and HCO have cross-reactivity; NOR is capable of reducing O2 and HCO is capable of reducing NO, although less efficiently.8–10 Not all members of the HCO superfamily are capable of NO reduction. The oxidase from bovine heart has no NO activity.11 In contrast, the ba3 and caa3 type oxidases from Thermus thermophilus9 and the cbb3 type oxidase from Pseudomonas stutzeri10 are capable of catalytically reducing NO to N2O, with the cbb3 oxidase being ~2 orders of magnitude faster than their oxidase counterparts. Although X-ray crystal structures are available5 for the oxidase from bovine heart,12,13 the aa3 type from Paracoccus denitrificans14,15 and Rhodobacter sphaeroides,16 the ba3 type from Thermus thermophilus,17 and the bo3 type ubiquinol oxidase from Escherichia coli,18 there is still no crystal structure for the cbb3 type oxidase, which from sequence alignments are predicted to be the most structurally similar oxidase to NOR.19 These crystal structures show the catalytic core of HCOs to contain a low-spin heme center and a high-spin heme/CuB binuclear center, (cytochrome c oxidase, CcO, also a member of the HCO superfamily, contains an additional binuclear CuA center). The CuB center consists of three strictly conserved His which bind a central Cu ion. One of these His is covalently attached to a strictly conserved Tyr to form a His-Tyr crosslink that is thought to play both a structural and functional role in O2 reduction.20 Another important characteristic of the heme-copper active site is the spin-coupled nature of the center in the oxidized or met form (i.e., CuII-FeIII) of the protein. Spin-coupling is thought to be provided by a bridging ligand between the two metal centers.21–25 Currently, no crystal or solution structure exists for bacterial NOR but sequence alignments and structural modeling have shown NORs to be structurally homologous to subunit I of HCOs.6,7 All six His metal ligands to the active site of HCO (two to the low-spin heme, one to the high-spin heme and three to CuB) are conserved in NOR at the same predicted positions in the 12 trans-membrane helices of HCO. The major structural difference between HCO and NOR is the replacement of a copper binding site (CuB) with a non-heme iron binding site (FeB) in NOR. Additionally, several conserved glutamates have been identified in the vicinity of the catalytic heme/non-heme FeB site of NOR which are predicted to play a role in iron binding and/or catalysis.26 Because of the importance of HCO to respiration, the structural similarities between NOR and HCO, and the importance of NOR to the denitrification cycle, it is important to reach a clear understanding of the structure and function of these vital enzymes. Biochemical, spectroscopic, and X-ray crystallographic studies of both HCOs12–15,27–37 and NORs38–49 are sometimes hampered by their size and complexity. Both are membrane-bound proteins containing several different metal cofactors, making their isolation difficult and spectroscopic characterization complicated (e.g., UV-vis and MCD spectra of the heme-copper center in HCO are masked by the low-spin heme center). These difficulties call upon the need for innovative ways to gain insight into the structure-function relationship of these heme proteins.

Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase

The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in ~13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site.

Active-Site Models of Bacterial Nitric Oxide Reductase Featuring Tris-Histidyl and Glutamic Acid Mimics: Influence of a Carboxylate Ligand on FeB Binding and the Heme Fe/FeB Redox Potential

Active-site models of bacterial nitric oxide reductase (NOR) featuring a heme Fe and a trisimidazole- and glutaric acid-bound non-heme Fe (Fe(B)) have been synthesized. These models closely replicate the proposed active site of native NORs. Examination of these models shows that the glutamic acid mimic is required for both Fe(B) retention in the distal binding site and proper modulation of the redox potentials of both the heme and non-heme Fe's.