Thioester synthesis by a designed nickel enzyme models prebiotic energy conversion.

The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the NiI species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.

Controlled Protonation of [2Fe-2S] Leading to MitoNEET Analogues and Concurrent Cluster Modification.

MitoNEET, a key regulatory protein in mitochondrial energy metabolism, exhibits a uniquely ligated [2Fe-2S] cluster with one histidine and three cysteines. This unique cluster has been postulated to sense the redox environment and release Fe-S cofactors under acidic pH. Reported herein is a synthetic system that shows how [2Fe-2S] clusters react with protons and rearrange their coordination geometry. The low-temperature stable, site-differentiated clusters [Fe2S2(SPh)3(CF3COO)]2- and [Fe2S2(SPh)3(py)]- have been prepared via controlled protonation below -35 °C and characterized by NMR, UV-vis, and X-ray absorption spectroscopy. Both complexes exhibit anodically shifted redox potentials compared to [Fe2S2(SPh)4]2- and convert to [Fe4S4(SPh)4]2- upon warming to room temperature. The current study provides insight into how mitoNEET releases its [2Fe-2S] in response to highly tuned acidic conditions, the chemistry of which may have further implications in Fe-S biogenesis.

A Biochemical Nickel(I) State Supports Nucleophilic Alkyl Addition: A Roadmap for Methyl Reactivity in Acetyl Coenzyme A Synthase.

Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in global energy conversion reactions, with significant contributions to carbon-centered processes. These enzymes are implied to cycle through a series of nickel-based organometallic intermediates during catalysis, though identification of these intermediates remains challenging. In this work, we have developed and characterized a nickel-containing metalloprotein that models the methyl-bound organometallic intermediates proposed in the native enzymes. Using a nickel(I)-substituted azurin mutant, we demonstrate that alkyl binding occurs via nucleophilic addition of methyl iodide as a methyl donor. The paramagnetic NiIII-CH3 species initially generated can be rapidly reduced to a high-spin NiII-CH3 species in the presence of exogenous reducing agent, following a reaction sequence analogous to that proposed for ACS. These two distinct bioorganometallic species have been characterized by optical, EPR, XAS, and MCD spectroscopy, and the overall mechanism describing methyl reactivity with nickel azurin has been quantitatively modeled using global kinetic simulations. A comparison between the nickel azurin protein system and existing ACS model compounds is presented. NiIII-CH3 Az is only the second example of two-electron addition of methyl iodide to a NiI center to give an isolable species and the first to be formed in a biologically relevant system. These results highlight the divergent reactivity of nickel across the two intermediates, with implications for likely reaction mechanisms and catalytically relevant states in the native ACS enzyme.

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center Within a Peptide Scaffold.

A disulfide-bridged peptide containing two Ni2+ binding sites based on the nickel superoxide dismutase protein, {Ni2(SODmds)}, has been prepared. At physiological pH (7.4) it was found that the metal sites are mononuclear with a square planar NOS2 coordination environment with the two sulfur-based ligands derived from cysteinate residues, the nitrogen ligand derived from the amide backbone and a water ligand. Furthermore, S K-edge X-ray absorption spectroscopy indicated that the two cysteinate sulfur atoms ligated to nickel are each protonated. Elevation of the pH to 9.6 results in the deprotonation of the cysteinate sulfur atoms, and yields a binuclear, cysteinate bridged Ni22+ center with each nickel contained in a distorted square planar geometry. At both pH = 7.4 and 9.6 the nickel sites are moderately air sensitive, yielding intractable oxidation products. However, at pH = 9.6 {Ni2(SODmds)} reacts with O2 at an ~3.5-fold faster rate than at pH = 7.4. Electronic structure calculations indicate the reduced reactivity at pH = 7.4 is a result of a reduction in S(3p) character and deactivation of the nucleophilic frontier molecular orbitals upon cysteinate sulfur protonation.

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2.

The critical nature of the copper transporter 1 (Ctr1) in human health has spurred investigation of Ctr1 structure and function. Ctr1 specifically transports Cu(I), the reduced form of copper, across the plasma membrane. Thus, extracellular Cu(II) must be reduced prior to transport. Unlike yeast Ctr1, mammalian Ctr1 does not rely on any known mammalian reductase. Previous spectroscopic studies of model peptides indicate that human Ctr1 could serve as both copper reductase and transporter. Ctr1 peptides bind Cu(II) at an amino terminal high-affinity Cu(II), Ni(II) ATCUN site. Ascorbate-dependent reduction of the Cu(II)-ATCUN complex is possible by virtue of an adjacent HH (bis-His), as this bis-His motif and one methionine ligand constitute a high affinity Ctr1 Cu(I) binding site. Here, we synthetically varied the distance between the ATCUN and bis-His motifs in a series of peptides based on the human Ctr1 amino terminal, with the general sequence MDHAnHHMGMSYMDS, where n=0-4. We tested the ability of each peptide to reduce Cu(II) with ascorbate and stabilize Cu(I) under ambient conditions (20% O2). This study reveals that significant differences in coordination structure and chemical behavior with ascorbate and O2 result from changes in the sequence proximity of ATCUN and bis-His. Peptides that deviate from the native Ctr1 pattern were less effective at forming stable Cu(I)-peptide complexes and/or resulted in O2-dependent oxidative damage to the peptide.

Model Peptide Studies Reveal a Mixed Histidine-Methionine Cu(I) Binding Site at the N-Terminus of Human Copper Transporter 1.

Copper is a vital metal cofactor in enzymes that are essential to myriad biological processes. Cellular acquisition of copper is primarily accomplished through the Ctr family of plasma membrane copper transport proteins. Model peptide studies indicate that the human Ctr1 N-terminus binds to Cu(II) with high affinity through an amino terminal Cu(II), Ni(II) (ATCUN) binding site. Unlike typical ATCUN-type peptides, the Ctr1 peptide facilitates the ascorbate-dependent reduction of Cu(II) bound in its ATCUN site by virtue of an adjacent HH (bis-His) sequence in the peptide. It is likely that the Cu(I) coordination environment influences the redox behavior of Cu bound to this peptide; however, the identity and coordination geometry of the Cu(I) site has not been elucidated from previous work. Here, we show data from NMR, XAS, and structural modeling that sheds light on the identity of the Cu(I) binding site of a Ctr1 model peptide. The Cu(I) site includes the same bis-His site identified in previous work to facilitate ascorbate-dependent Cu(II) reduction. The data presented here are consistent with a rational mechanism by which Ctr1 provides coordination environments that facilitate Cu(II) reduction prior to Cu(I) transport.

Adiabaticity of the proton-coupled electron-transfer step in the reduction of superoxide effected by nickel-containing superoxide dismutase metallopeptide-based mimics.

Nickel-containing superoxide dismutases (NiSODs) are bacterial metalloenzymes that catalyze the disproportionation of O2(-). These enzymes take advantage of a redox-active nickel cofactor, which cycles between the Ni(II) and Ni(III) oxidation states, to catalytically disprotorptionate O2(-). The Ni(II) center is ligated in a square planar N2S2 coordination environment, which, upon oxidation to Ni(III), becomes five-coordinate following the ligation of an axial imidazole ligand. Previous studies have suggested that metallopeptide-based mimics of NiSOD reduce O2(-) through a proton-coupled electron transfer (PCET) reaction with the electron derived from a reduced Ni(II) center and the proton from a protonated, coordinated Ni(II)-S(H(+))-Cys moiety. The current work focuses on the O2(-) reduction half-reaction of the catalytic cycle. In this study we calculate the vibronic coupling between the reactant and product diabatic surfaces using a semiclassical formalism to determine if the PCET reaction is proceeding through an adiabatic or nonadiabatic proton tunneling process. These results were then used to calculate H/D kinetic isotope effects for the PCET process. We find that as the axial imidazole ligand becomes more strongly associated with the Ni(II) center during the PCET reaction, the reaction becomes more nonadiabatic. This is reflected in the calculated H/D KIEs, which moderately increase as the reaction becomes more nonadiabatic. Furthermore, the results suggest that as the axial ligand becomes less Lewis basic the observed reaction rate constants for O2(-) reduction should become faster because the reaction becomes more adiabatic. These conclusions are in-line with experimental observations. The results thus indicate that variations in the axial donor's ability to coordinate to the nickel center of NiSOD metallopeptide-based mimics will strongly influence the fundamental nature of the O2(-) reduction process.

A nickel phosphine complex as a fast and efficient hydrogen production catalyst.

Here we report the electrocatalytic reduction of protons to hydrogen by a novel S2P2 coordinated nickel complex, [Ni(bdt)(dppf)] (bdt = 1,2-benzenedithiolate, dppf = 1,1'-bis(diphenylphosphino)ferrocene). The catalysis is fast and efficient with a turnover frequency of 1240 s(-1) and an overpotential of only 265 mV for half activity at low acid concentrations. Furthermore, catalysis is possible using a weak acid, and the complex is stable for at least 4 h in acidic solution. Calculations of the system carried out at the density functional level of theory (DFT) are consistent with a mechanism for catalysis in which both protonations take place at the nickel center.

Cysteinate protonation and water hydrogen bonding at the active-site of a nickel superoxide dismutase metallopeptide-based mimic: implications for the mechanism of superoxide reduction.

Nickel-containing superoxide dismutase (NiSOD) is a mononuclear cysteinate-ligated nickel metalloenzyme that catalyzes the disproportionation of superoxide into dioxygen and hydrogen peroxide by cycling between Ni(II) and Ni(III) oxidation states. All of the ligating residues to nickel are found within the first six residues from the N-terminus, which has prompted several research groups to generate NiSOD metallopeptide-based mimics derived from the first several residues of the NiSOD sequence. To assess the viability of using these metallopeptide-based mimics (NiSOD maquettes) to probe the mechanism of SOD catalysis facilitated by NiSOD, we computationally explored the initial step of the O2(-) reduction mechanism catalyzed by the NiSOD maquette {Ni(II)(SOD(m1))} (SOD(m1) = HCDLP CGVYD PA). Herein we use spectroscopic (S K-edge X-ray absorption spectroscopy, electronic absorption spectroscopy, and circular dichroism spectroscopy) and computational techniques to derive the detailed active-site structure of {Ni(II)(SOD(m1))}. These studies suggest that the {Ni(II)(SOD(m1))} active-site possesses a Ni(II)-S(H(+))-Cys(6) moiety and at least one associated water molecule contained in a hydrogen-bonding interaction to the coordinated Cys(2) and Cys(6) sulfur atoms. A computationally derived mechanism for O2(-) reduction using the formulated active-site structure of {Ni(II)(SOD(m1))} suggests that O2(-) reduction takes place through an apparent initial outersphere hydrogen atom transfer (HAT) from the Ni(II)-S(H(+))-Cys(6) moiety to the O2(-) molecule. It is proposed that the water molecule aids in driving the reaction forward by lowering the Ni(II)-S(H(+))-Cys(6) pK(a). Such a mechanism is not possible in NiSOD itself for structural reasons. These results therefore strongly suggest that maquettes derived from the primary sequence of NiSOD are mechanistically distinct from NiSOD itself despite the similarities in the structure and physical properties of the metalloenzyme vs the NiSOD metallopeptide-based models.

Insight into the structure and mechanism of nickel-containing superoxide dismutase derived from peptide-based mimics.

Nickel superoxide dismutase (NiSOD) is a nickel-containing metalloenzyme that catalyzes the disproportionation of superoxide through a ping-pong mechanism that relies on accessing reduced Ni(II) and oxidized Ni(III) oxidation states. NiSOD is the most recently discovered SOD. Unlike the other known SODs (MnSOD, FeSOD, and (CuZn)SOD), which utilize "typical" biological nitrogen and oxygen donors, NiSOD utilizes a rather unexpected ligand set. In the reduced Ni(II) oxidation state, NiSOD utilizes nitrogen ligands derived from the N-terminal amine and an amidate along with two cysteinates sulfur donors. These are unusual biological ligands, especially for an SOD: amine and amidate donors are underrepresented as biological ligands, whereas cysteinates are highly susceptible to oxidative damage. An axial histidine imidazole binds to nickel upon oxidation to Ni(III). This bond is long (2.3-2.6 Å) owing to a tight hydrogen-bonding network. All of the ligating residues to Ni(II) and Ni(III) are found within the first 6 residues from the NiSOD N-terminus. Thus, small nickel-containing metallopeptides derived from the first 6-12 residues of the NiSOD sequence can reproduce many of the properties of NiSOD itself. Using these nickel-containing metallopeptide-based NiSOD mimics, we have shown that the minimal sequence needed for nickel binding and reproduction of the structural, spectroscopic, and functional properties of NiSOD is H2N-HCXXPC. Insight into how NiSOD avoids oxidative damage has also been gained. Using small NiN2S2 complexes and metallopeptide-based mimics, it was shown that the unusual nitrogen donor atoms protect the cysteinates from oxidative damage (both one-electron oxidation and oxygen atom insertion reactions) by fine-tuning the electronic structure of the nickel center. Changing the nitrogen donor set to a bis-amidate or bis-amine nitrogen donor led to catalytically nonviable species owing to nickel-cysteinate bond oxidative damage. Only the amine/amidate nitrogen donor atoms within the NiSOD ligand set produce a catalytically viable species. These metallopeptide-based mimics have also hinted at the detailed mechanism of SOD catalysis by NiSOD. One such aspect is that the axial imidazole likely remains ligated to the Ni center under rapid catalytic conditions (i.e., high superoxide loads). This reduces the degree of structural rearrangement about the nickel center, leading to higher catalytic rates. Metallopeptide-based mimics have also shown that, although an axial ligand to Ni(III) is required for catalysis, the rates are highest when this is a weak interaction, suggesting a reason for the long axial His-Ni(III) bond found in NiSOD. These mimics have also suggested a surprising mechanistic insight: O2(-) reduction via a "H(•)" tunneling event from a R-S(H(+))-Ni(II) moiety to O2(-) is possible. The importance of this mechanism in NiSOD has not been verified.

Transformation of a mononitrosyl iron complex to a [2Fe-2S] cluster by a cysteine analogue.

Reversible modification of iron-sulfur clusters by nitric oxide acts as a genetic switch in a group of regulatory proteins. While the conversion of [Fe-S] clusters to iron-nitrosyls has been widely studied in the past, little is known about the reverse process, the repair of [Fe-S] clusters. Reported here is a system in which a mononitrosyl iron complex (MNIC), (PPN)[Fe(S(t)Bu)3(NO)] (1), is converted to a [2Fe-2S] cluster, (PPN)2[Fe2S2(SCH2CH2C(O)OMe)4] (2). This conversion requires only the addition of a cysteine analogue, 3-mercaptomethylpropionate (MMP), at room temperature without the need for any other reagents. The identity of 2 was confirmed spectroscopically, chemically, crystallographically, and analytically. Mass spectrometry and (34)S labeling studies support that the bridging sulfides in 2 derive from the added MMP, the cysteine analogue. The NO lost during the conversion of 1 to 2 is trapped in a dinitrosyl iron side product, (PPN)[Fe(SCH2CH2C(O)OMe)2(NO)2] (4). The present system implies that MNICs are likely intermediates in the repair of NO-damaged [2Fe-2S] clusters and that cysteine is a viable molecule responsible for the destabilization of MINCs and the formation of [2Fe-2S] clusters.

Dioxygen and superoxide stability of metallopeptide based mimics of nickel containing superoxide dismutase: the influence of amine/amidate vs. bis-amidate ligation.

Nickel containing superoxide dismutase (NiSOD) is a metalloenzyme that catalyzes the disproportionation of O2(-). In its reduced state, the Ni(II) ion is coordinated by two cis-cysteinates, an amine nitrogen and an amidate nitrogen atom. It thus bears a resemblance to the distal bis-cysteinate bis-amidate ligated nickel center of acetyl coenzyme A synthase. Using metallopeptide based NiSOD mimics derived from the first 12 residues of the NiSOD sequence we demonstrate that altering the primary coordination sphere from a bis-thiolate amine/amidate motif to a bis-thiolate bis-amidate motif changes the O2 and ROS stability of the metallopeptide. Using FT-IR, ESI-MS and S K-edge XAS we show that the bis-amidate bis-thiolate ligated metallopeptide {Ni(II)(SOD(m1)-Ac)} (SOD(m1)-Ac=AcHN-HCDLPCGVYSPA-COOH) undergoes oxidation at one thiolate ligand in the presence of O2, converting it into a coordinated sulfinate. Upon exposure of {Ni(II)(SOD(m1)-Ac)} to O2(-) the metallopeptide undergoes extensive sulfur oxidation. This can be contrasted with the unacylated metallopeptide {Ni(II)(SOD(m1))} which does not undergo sulfur based oxidation under these conditions. The biological implications of these findings are discussed.

Dioxygen mediated conversion of {Fe(NO)2}9 dinitrosyl iron complexes to Roussin's red esters.

The reactivity of a series of {Fe(NO)2}(9) dinitrosyl iron complexes bearing thiolate ligands with molecular oxygen is reported. These reactions result in the formation of the corresponding Roussin's red esters along with thiolate oxidation. This reactivity is contrasted with that previously reported for {Fe(NO)2}(10) complexes.

Sequential oxidations of thiolates and the cobalt metallocenter in a synthetic metallopeptide: implications for the biosynthesis of nitrile hydratase.

Cobalt nitrile hydratases (Co-NHase) contain a catalytic cobalt(III) ion coordinated in an N2S3 first coordination sphere composed of two amidate nitrogens and three cysteine-derived sulfur donors: a thiolate (-SR), a sulfenate (-S(R)O(-)), and a sulfinate (-S(R)O2(-)). The sequence of biosynthetic reactions that leads to the post-translational oxidations of the metal and the sulfur ligands is unknown, but the process is believed to be initiated directly by oxygen. Herein we utilize cobalt bound in an N2S2 first coordination sphere by a seven amino acid peptide known as SODA (ACDLPCG) to model this oxidation process. Upon exposure to oxygen, Co-SODA is oxidized in two steps. In the first fast step (seconds), magnetic susceptibility measurements demonstrated that the metallocenter remains paramagnetic, that is, Co(2+), and sulfur K-edge X-ray absorption spectroscopy (XAS) is used to show that one of the thiolates is oxidized to sulfinate. In a second process on a longer time scale (hours), magnetic susceptibility measurements and Co K-edge XAS show that the metal is oxidized to Co(3+). Unlike other model complexes, additional slow oxidation of the second thiolate in Co-SODA is not observed, and a catalytically active complex is never formed. The likely reason is the absence of the axial thiolate ligand. In essence, the reactivity of Co-SODA can be described as between previously described models which either quickly convert to final product or are stable in air, and it offers a first glimpse into a possible oxidation pathway for nitrile hydratase biosynthesis.

Use of a metallopeptide-based mimic provides evidence for a proton-coupled electron-transfer mechanism for superoxide reduction by nickel-containing superoxide dismutase.

Sneaky little SOD! A metallopeptide-based mimic of nickel-containing superoxide dismutase was used to probe the mechanism of superoxide reduction by the metalloenzyme. Kinetic studies suggest a proton-coupled electron-transfer mechanism; large H/D kinetic isotope effects (KIE) are observed. XAS studies suggest the transferred H-atom is in the form of a Ni(II) -S(H)-Cys moiety.

Influence of sequential thiolate oxidation on a nitrile hydratase mimic probed by multiedge X-ray absorption spectroscopy.

Nitrile hydratases (NHases) are Fe(III)- and Co(III)-containing hydrolytic enzymes that convert nitriles into amides. The metal-center is contained within an N(2)S(3) coordination motif with two post-translationally modified cysteinates contained in a cis arrangement, which have been converted into a sulfinate (R-SO(2)(-)) and a sulfenate (R-SO(-)) group. Herein, we utilize Ru L-edge and ligand (N-, S-, and P-) K-edge X-ray absorption spectroscopies to probe the influence that these modifications have on the electronic structure of a series of sequentially oxidized thiolate-coordinated Ru(II) complexes ((bmmp-TASN)RuPPh(3), (bmmp-O(2)-TASN)RuPPh(3), and (bmmp-O(3)-TASN)RuPPh(3)). Included is the use of N K-edge spectroscopy, which was used for the first time to extract N-metal covalency parameters. We find that upon oxygenation of the bis-thiolate compound (bmmp-TASN)RuPPh(3) to the sulfenato species (bmmp-O(2)-TASN)RuPPh(3) and then to the mixed sulfenato/sulfinato speices (bmmp-O(3)-TASN)RuPPh(3) the complexes become progressively more ionic, and hence the Ru(II) center becomes a harder Lewis acid. These findings are reinforced by hybrid DFT calculations (B(38HF)P86) using a large quadruple-ζ basis set. The biological implications of these findings in relation to the NHase catalytic cycle are discussed in terms of the creation of a harder Lewis acid, which aids in nitrile hydrolysis.

Modeling the geometric, electronic, and redox properties of iron(III)-containing amphiphiles with asymmetric [NN'O] headgroups.

Two iron(III)-containing amphiphiles 1 and 2 have been synthesized with the [NN'O] ligands HL(tBu-ODA) (2-((octadecyl(pyridin-2-ylmethyl)amino)methyl)-4,6-di-tert-butylphenol) and HL(I-ODA) (2-((octadecyl(pyridin-2-ylmethyl)amino)methyl)-4,6-diiodophenol), respectively. Compound 1 is monometallic, whereas EXAFS data suggest that 2 is a mixture of mono- and bimetallic species. The archetypical [Fe(III)(L(NN'O))(2)](+) complexes 3-9 have been isolated and characterized in order to understand the geometric, electronic, and redox properties of the amphiphiles. Preference for a monometallic or bimetallic nuclearity is dependent on (i) the nature of the solvent used for synthesis and (ii) the type of the substituent in the phenol moiety. In methanol, the tert-butyl-, methoxy-, and chloro-substituted 3, 4, and 5 are monometallic species, whereas the bromo- and iodo-substituted 6 and 7 form bimetallic complexes taking advantage of stabilizing methoxo bridges generated by solvent deprotonation. In dichloromethane, the bromo- and iodo-substituted 8 and 9 are monometallic species; however, these species favor meridional coordination in opposition to the facial coordination observed for the tert-butyl- and methoxy-substituted compounds. Molecular structures for species 5, 7, 8, and 9 have been solved by X-ray diffraction. Furthermore, the electronic spectrum of the amphiphile 1 was expected to be similar to those of facial/cis archetypes with similar substituents, but close resemblance was observed with the profile for those meridional/cis species, suggesting a similar coordination mode. This trend is discussed based on DFT calculations, where preference for the meridional/cis coordination mode appears related to the presence of tertiary amine nitrogen on the ligand, as when a long alkyl chain is attached to the [NN'O] headgroup.

Phenol nitration induced by an {Fe(NO)2}(10) dinitrosyl iron complex.

Cellular dinitrosyl iron complexes (DNICs) have long been considered NO carriers. Although other physiological roles of DNICs have been postulated, their chemical functionality outside of NO transfer has not been demonstrated thus far. Here we report the unprecedented dioxygen reactivity of a N-bound {Fe(NO)(2)}(10) DNIC, [Fe(TMEDA)(NO)(2)] (1). In the presence of O(2), 1 becomes a nitrating agent that converts 2,4,-di-tert-butylphenol to 2,4-di-tert-butyl-6-nitrophenol via formation of a putative iron-peroxynitrite [Fe(TMEDA)(NO)(ONOO)] (2) that is stable below -80 °C. Iron K-edge X-ray absorption spectroscopy on 2 supports a five-coordinated metal center with a bound peroxynitrite in a cyclic bidentate fashion. The peroxynitrite ligand of 2 readily decays at increased temperature or under illumination. These results suggest that DNICs could have multiple physiological or deleterious roles, including that of cellular nitrating agents.

Use of metallopeptide based mimics demonstrates that the metalloprotein nitrile hydratase requires two oxidized cysteinates for catalytic activity.

Nitrile hydratases (NHases) are non-heme Fe(III) or non-corrin Co(III) containing metalloenzymes that possess an N(2)S(3) ligand environment with nitrogen donors derived from amidates and sulfur donors derived from cysteinates. A closely related enzyme is thiocyanate hydrolase (SCNase), which possesses a nearly identical active-site coordination environment as CoNHase. These enzymes are redox inactive and perform hydrolytic reactions; SCNase hydrolyzes thiocyanate anions while NHase converts nitriles into amides. Herein an active CoNHase metallopeptide mimic, [Co(III)NHase-m1] (NHase-m1 = AcNH-CCDLP-CGVYD-PA-COOH), that contains Co(III) in a similar N(2)S(3) coordination environment as CoNHase is reported. [Co(III)NHase-m1] was characterized by electrospray ionization-mass spectrometry (ESI-MS), gel-permeation chromatography (GPC), Co K-edge X-ray absorption spectroscopy (Co-S: 2.21 Å; Co-N: 1.93 Å), vibrational, and optical spectroscopies. We find that [Co(III)NHase-m1] will perform the catalytic conversion of acrylonitrile into acrylamide with up to 58 turnovers observed after 18 h at 25 °C (pH 8.0). FTIR data used in concert with calculated vibrational data (mPWPW91/aug-cc-TZVPP) demonstrates that the active form of [Co(III)NHase-m1] has a ligated SO(2) (ν = 1091 cm(-1)) moiety and a ligated protonated SO(H) (ν = 928 cm(-1)) moiety; when only one oxygenated cysteinate ligand (i.e., a mono-SO(2) coordination motif) or the bis-SO(2) coordination motif are found within [Co(III)NHase-m1] no catalytic activity is observed. Calculations of the thermodynamics of ligand exchange (B3LYP/aug-cc-TZVPP) suggest that the reason for this is that the SO(2)/SO(H) equatorial ligand motif promotes both water dissociation from the Co(III)-center and nitrile coordination to the Co(III)-center. In contrast, the under- or overoxidized motifs will either strongly favor a five coordinate Co(III)-center or strongly favor water binding to the Co(III)-center over nitrile binding.

Bisamidate and mixed amine/amidate NiN2S2 complexes as models for nickel-containing acetyl coenzyme A synthase and superoxide dismutase: an experimental and computational study.

The distal nickel site of acetyl-CoA synthase (Ni(d)-ACS) and reduced nickel superoxide dismutase (Ni-SOD) display similar square-planar Ni(II)N(2)S(2) coordination environments. One difference between these two sites, however, is that the nickel ion in Ni-SOD contains a mixed amine/amidate coordination motif while the Ni(d) site in Ni-ACS contains a bisamidate coordination motif. To provide insight into the consequences of the different coordination environments on the properties of the Ni ions, we systematically examined two square-planar Ni(II)N(2)S(2) complexes, one with bisthiolate-bisamidate ligation (Et(4)N)(2)(Ni(L1)).2H(2)O (2) [H(4)L1 = N-(2-mercaptoacetyl)-N'-(2-mercaptoethyl)glycinamide] and another with bisthiolate-amine/amidate ligation K(Ni(HL2)) (3) [H(4)L2 = N-(2''-mercaptoethyl)-2-((2'-mercaptoethyl)amino)acetamide]. Although these two complexes differ only by a single amine versus amidate ligand, their chemical properties are quite different. The stronger in-plane ligand field in the bisamidate complex (Ni(II)(L1))(2-) (2) results in an increase in the energies of the d --> d transitions and a considerably more negative oxidation potential. Furthermore, while the bisamidate complex (Ni(II)(L1))(2-) (2) readily forms a trinuclear species (Et(4)N)(2)({Ni(L1)}(2)Ni).H(2)O (1) and reacts rapidly with O(2), presumably via sulfoxidation, the mixed amine/amidate complex (Ni(II)(HL2))(-) (3) remains monomeric and is stable for days in air. Interestingly, the Ni(III) species of the bisamidate complex formed by chemical oxidation with I(2) can be detected by electron paramagnetic resonance (EPR) spectroscopy while the mixed amine/amidate complex immediately decomposes upon oxidation. To explain these experimentally observed properties, we performed S K-edge X-ray absorption spectroscopy and low-temperature (77 K) electronic absorption measurements as well as both hybrid density functional theory (hybrid-DFT) and spectroscopy oriented configuration interaction (SORCI) calculations. These studies demonstrate that the highest occupied molecular orbital (HOMO) of the bisamidate complex (Ni(II)(L1))(2-) (2) has more Ni character and is significantly destabilized relative to the mixed amine/amidate complex (Ni(II)(HL2))(-) (3) by approximately 6.2 kcal mol(-1). The consequence of this destabilization is manifested in the nucleophilic activation of the doubly filled HOMO, which makes (Ni(II)(L1))(2-) (2) significantly more reactive toward electrophiles such as O(2).