Bacterial nitric oxide reductase (NorBC) models employing click chemistry.

Bacterial NO Reductase (NorBC or cNOR) is a membrane-bound enzyme found in denitrifying bacteria that catalyzes the two-electron reduction of NO to N2O and water. The mechanism by which NorBC operates is highly debated, due to the fact that this enzyme is difficult to work with, and no intermediates of the NO reduction reaction could have been identified so far. The unique active site of NorBC consists of a heme b3/non-heme FeB diiron center. Synthetic model complexes provide the opportunity to obtain insight into possible mechanistic alternatives for this enzyme. In this paper, we present three new synthetic model systems for NorBC, consisting of a tetraphenylporphyrin-derivative clicked to modified BMPA-based ligands (BMPA = bis(methylpyridyl)amine) that model the non-heme site in the enzyme. These complexes have been characterized by EPR, IR and UV-Vis spectroscopy. The reactivity with NO was then investigated, and it was found that the complex with the BMPA-carboxylate ligand as the non-heme component has a very low affinity for NO at the non-heme iron site. If the carboxylate functional group is replaced with a phenolate or pyridine group, reactivity is restored and formation of a diiron dinitrosyl complex was observed. Upon one-electron reduction of the nitrosylated complexes, following the semireduced pathway for NO reduction, formation of dinitrosyl iron complexes (DNICs) was observed in all three cases, but no N2O could be detected.

DFT insights into the mechanism of O2 activation catalyzed by a structural and functional model of cysteine dioxygenase with tris(2-pyridyl)methane-based ligand architecture.

Cysteine dioxygenation is an important step in the metabolism of toxic L-cysteine (Cys) in the human body, carried out by cysteine dioxygenase enzyme (CDO). The disruption of this process is found to elicit neurological health issues. This work reports a computational investigation of mechanistic aspects of this reaction, using a recently reported tris(2-pyridyl)methane-based biomimetic model complex of CDO. The computed results indicate that, the initial SO2 bond formation process is the slowest step in the S-dioxygenation process, possessing an activation barrier of 12.7 kcal/mol. The remaining steps were found to be downhill requiring very small activation energies. The transition states were found to undergo spin crossover between triplet and quintet states, while the singlet surface remained unstable throughout the entire reaction. In essence, the mechanistic scheme and multistate reactivity pattern together with the relatively small computed rate-limiting activation barrier as well as the exothermic formation energy demonstrate that the model complex is an efficient biomimetic CDO model. In addition, the study also substantiates the involvement of Fe(IV)oxido intermediates in the mechanism of S-dioxygenation by the chosen model complex. The insights derived from the O2 activation process might pave way for development of more accurate CDO model catalysts that might be capable of even more efficiently mimicking the geometric, spectroscopic and functional features of the CDO enzyme.

Synthesis and characterization of a model complex for flavodiiron NO reductases that stabilizes a diiron mononitrosyl complex.

Flavodiiron NO reductases (FNORs) are important enzymes in microbial pathogenesis, as they equip microbes with resistance to the human immune defense agent nitric oxide (NO). DFT calculations predict that a network of second coordination sphere (SCS) hydrogen bonds is critical for the key NN coupling step in the NO reduction reaction catalyzed by FNORs. In this study, we report the synthesis of a model complex of FNORs with pendant hydrogen bond donors. For this purpose, the ligand H[BPMP] (= 2,6-bis[[bis(2-pyridylmethyl)amino]methyl]-4-methylphenol) was modified with two amide groups in the SCS. Reaction of the precursor complex [Fe2(BPMP(NHCOtBu)2)(OAc)](OTf)2 (1) (OTf- = triflate anion) with NO in the presence of base led to the surprising isolation of a diiron mononitrosyl complex, [Fe2(BPMP(NHCOtBu)(NCOtBu))(OAc)(NO)](OTf) (2) and a triiron decomposition product, [Fe3(BPMP(NHCOtBu)2)(OAc)2(µ-O)2(ONO)](OTf) (3), which were both structurally characterized. Complex 2 models the corresponding mononitrosyl adduct in FNORs. This result points towards a strategy that can be used to stabilize mononitrosyl diiron complexes, using the SCS.

Influence of the amine donor on hybrid guanidine-stabilized Bis(µ-oxido) dicopper(III) complexes and their tyrosinase-like oxygenation activity towards polycyclic aromatic alcohols.

The tyrosinase-like activity of hybrid guanidine-stabilized bis(µ-oxido) dicopper(III) complexes [Cu2(µ-O)2(L)2](X)2 (L = 2-{2-((Diethylamino)methyl)phenyl}-1,1,3,3-tetramethylguanidine (TMGbenzNEt2, L2) and 2-{2-((Di-isopropylamino)methyl)phenyl}-1,1,3,3-tetramethylguanidine (TMGbenzNiPr2, L3); X = PF6-, BF4-, CF3SO3-) is described. New aromatic hybrid guanidine amine ligands were developed with varying amine donor function. Their copper(I) complexes were analyzed towards their ability to activate dioxygen in the presence of different weakly coordinating anions. The resulting bis(µ-oxido) species were characterized at low temperatures by UV/Vis and resonance Raman spectroscopy, cryo-ESI mass spectrometry and density functional theory calculations. Small structural changes in the ligand sphere were found to influence the characteristic ligand-to-metal charge transfer (LMCT) features of the bis(µ-oxido) species, correlating a redshift in the UV/Vis spectrum with weaker N-donor function of the ligand. DFT calculations elucidated the influence of the steric and electronic properties of the bis(µ-oxido) species leading to a higher twist of the Cu2O2 plane against the CuN2 plane and a stretching of the Cu2O2 core. Despite their moderate stability at -100 °C, the bis(µ-oxido) complexes exhibited a remarkable activity in catalytic oxygenation reactions of polycyclic aromatic alcohols. Further the selectivity of the catalyst in the hydroxylation reactions of challenging phenolic substrates is not changed despite an increasing shield of the reactive bis(µ-oxido) core. The generated quinones were found to form exclusively bent phenazines, providing a promising strategy to access tailored phenazine derivatives.

Using a monocopper-superoxo complex to prepare multicopper-peroxo species relevant to proposed enzyme intermediates.

With the goal of generating a (peroxo)tricopper species analogous to the Peroxy Intermediate proposed for multicopper oxidases, solutions of the copper-superoxide complex [K(Krypt)][LCuO2] (L = N,N'-bis(2,6-diisopropylphenyl)-2,6-pyridinedicarboxamide, Krypt = 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane) were reacted with the dicopper(I) complex [(TPBN)Cu2(MeCN)2][PF6]2 at -70 °C (TPBN = N,N,N',N'-tetrakis-(2-pyridylmethyl)-1,4-diaminobutane). A metastable intermediate formed, which on the basis of UV-vis, EPR, and resonance Raman spectroscopy was proposed to derive from reaction of two equivalents of the copper-superoxide with one equivalent of the dicopper(I) complex to yield a complex with two (peroxo)dicopper moieties rather than the desired (peroxo)tricopper PI model. A similar intermediate formed upon reaction of [K(Krypt)][LCuO2] with [(BPMA)Cu(MeCN)][PF6] (BPMA = N,N-bis(2-pyridylmethyl)-methyl-amine), which contained the same donor set as provided by TPBN. Comparison of resonance Raman data and consideration of structural preferences for LCuX species led to hypothesis of a µ-η1:η2-peroxo structure for both intermediates.

CaMn3IV O4 Cubane Models of the Oxygen-Evolving Complex: Spin Ground States S<9/2 and the Effect of Oxo Protonation.

We report the single crystal XRD and MicroED structure, magnetic susceptibility, and EPR data of a series of CaMn3IV O4 and YMn3IV O4 complexes as structural and spectroscopic models of the cuboidal subunit of the oxygen-evolving complex (OEC). The effect of changes in heterometal identity, cluster geometry, and bridging oxo protonation on the spin-state structure was investigated. In contrast to previous computational models, we show that the spin ground state of CaMn3IV O4 complexes and variants with protonated oxo moieties need not be S=9/2. Desymmetrization of the pseudo-C3 -symmetric Ca(Y)Mn3IV O4 core leads to a lower S=5/2 spin ground state. The magnitude of the magnetic exchange coupling is attenuated upon oxo protonation, and an S=3/2 spin ground state is observed in CaMn3IV O3 (OH). Our studies complement the observation that the interconversion between the low-spin and high-spin forms of the S2 state is pH-dependent, suggesting that the (de)protonation of bridging or terminal oxygen atoms in the OEC may be connected to spin-state changes.

Recent Advances in One-Electron-Oxidized CuII -Diphenoxide Complexes as Models of Galactose Oxidase: Importance of the Structural Flexibility in the Active Site.

The phenoxyl radical plays important roles in biological systems as cofactors in some metalloenzymes, such as galactose oxidase (GO) catalyzing oxidation of primary alcohols to give the corresponding aldehydes. Many metal(II)-phenoxyl radical complexes have hitherto been studied for understanding the detailed properties and reactivities of GO, and thus the nature of GO has gradually become clearer. However, the effects of the subtle geometric and electronic structural changes at the active site of GO, especially the structural change in the catalytic cycle and the effect of the second coordination sphere, have not been fully discussed yet. In this Review, we focus on further details of the model studies of GO and discuss the importance of the structural change at the active site of GO.

Nitric Oxide Generation On Demand for Biomedical Applications via Electrocatalytic Nitrite Reduction by Copper BMPA- and BEPA-Carboxylate Complexes.

Intravascular (IV) catheters are essential devices in the hospital that are used to monitor a patient's blood and for administering drugs or nutrients. However, IV catheters are also prone to blood clotting at the point of insertion and infection by formation of robust bacterial biofilms on their surface. Nitric oxide (NO) is ideally suited to counteract both of these problems, due to its antimicrobial properties and its ability to inhibit platelet activation/aggregation. One way to equip catheters with NO releasing properties is by electrocatalytic nitrite reduction to NO by copper complexes in a multi-lumen configuration. In this work, we systematically investigate six closely related Cu(II) BMPA- and BEPA-carboxylate complexes (BMPA = bis-(2-methylpyridyl)amine); BEPA = bis-(2-ethylpyridyl)amine), using carboxylate groups of different chain lengths. The corresponding Cu(II) complexes were characterized using UV-Vis, EPR spectroscopy, and X-ray crystallography. Using detailed cyclic voltammetry (CV) and bulk electrocatalyic studies (with real-time NO quantification), in aqueous buffer, pH 7.4, we are able to derive clear reactivity relations between the ligand structures of the complexes, their Faradaic efficiencies for NO generation, their turnover frequencies (TOFs), and their redox potentials. Our results show that the complex [Cu(BEPA-Bu)](OAc) is the best catalyst with a high Faradaic efficiency over large nitrite concentration ranges and the expected best tolerance to oxygen levels. For this species, the more positive redox potential suppresses NO disproportionation, which is a major Achilles heel of the (faster) catalysts with the more negative reduction potentials.

The Fe2 (NO)2 Diamond Core: A Unique Structural Motif In Non-Heme Iron-NO Chemistry.

Non-heme high-spin (hs) {FeNO}8 complexes have been proposed as important intermediates towards N2 O formation in flavodiiron NO reductases (FNORs). Many hs-{FeNO}8 complexes disproportionate by forming dinitrosyl iron complexes (DNICs), but the mechanism of this reaction is not understood. While investigating this process, we isolated a new type of non-heme iron nitrosyl complex that is stabilized by an unexpected spin-state change. Upon reduction of the hs-{FeNO}7 complex, [Fe(TPA)(NO)(OTf)](OTf) (1), the N-O stretching band vanishes, but no sign of DNIC or N2 O formation is observed. Instead, the dimer, [Fe2 (TPA)2 (NO)2 ](OTf)2 (2) could be isolated and structurally characterized. We propose that 2 is formed from dimerization of the hs-{FeNO}8 intermediate, followed by a spin state change of the iron centers to low-spin (ls), and speculate that 2 models intermediates in hs-{FeNO}8 complexes that precede the disproportionation reaction.

Oxygen activation by mononuclear Mn, Co, and Ni centers in biology and synthetic complexes.

The active sites of metalloenzymes that catalyze O2-dependent reactions generally contain iron or copper ions. However, several enzymes are capable of activating O2 at manganese or nickel centers instead, and a handful of dioxygenases exhibit activity when substituted with cobalt. This minireview summarizes the catalytic properties of oxygenases and oxidases with mononuclear Mn, Co, or Ni active sites, including oxalate-degrading oxidases, catechol dioxygenases, and quercetin dioxygenase. In addition, recent developments in the O2 reactivity of synthetic Mn, Co, or Ni complexes are described, with an emphasis on the nature of reactive intermediates featuring superoxo-, peroxo-, or oxo-ligands. Collectively, the biochemical and synthetic studies discussed herein reveal the possibilities and limitations of O2 activation at these three "overlooked" metals.

Hydrophilic Quaternary Ammonium-Group-Containing [FeFe]-Hydrogenase Models: Synthesis, Structures, and Electrocatalytic Hydrogen Production.

The first quaternary ammonium-group-containing [FeFe]-hydrogenase models [(µ-PDT)Fe2 (CO)4 {κ2 -(Ph2 P)2 N(CH2 )2 NMe2 BzBr}] (2; PDT=propanedithiolate) and [(µ-PDT)Fe2 (CO)4 {µ-(Ph2 P)2 N(CH2 )2 NMe2 BzBr}] (4) have been prepared by the quaternization of their precursors [(µ-PDT)Fe2 (CO)4 {κ2 -(Ph2 P)2 N(CH2 )2 NMe2 }] (1) and [(µ-PDT)Fe2 (CO)4 {µ-(Ph2 P)2 N(CH2 )2 NMe2 }] (3) with benzyl bromide in high yields. Although new complexes 1-4 have been fully characterized by spectroscopic and X-ray crystallographic studies, the chelated complexes 1 and 2 converted into their bridged isomers 3 and 4 at higher temperatures, thus demonstrating that these bridged isomers are thermodynamically favorable. An electrochemical study on hydrophilic models 2 and 4 in MeCN and MeCN/H2 O as solvents indicates that the reduction potentials are shifted to less-negative potentials as the water content increases. This outcome implies that both 2 and 4 are more easily reduced in the mixed MeCN/H2 O solvent than in MeCN. In addition, hydrophilic models 2 and 4 act as electrocatalysts and achieve higher icat /ip values and turnover numbers (TONs) in MeCN/H2 O as a solvent than in MeCN for the production of hydrogen from the weak acid HOAc.

Reduction potentials of heterometallic manganese-oxido cubane complexes modulated by redox-inactive metals.

Understanding the effect of redox-inactive metals on the properties of biological and heterogeneous water oxidation catalysts is important both fundamentally and for improvement of future catalyst designs. In this work, heterometallic manganese-oxido cubane clusters [MMn3O4] (M = Sr(2+), Zn(2+), Sc(3+), Y(3+)) structurally relevant to the oxygen-evolving complex (OEC) of photosystem II were prepared and characterized. The reduction potentials of these clusters and other related mixed metal manganese-tetraoxido complexes are correlated with the Lewis acidity of the apical redox-inactive metal in a manner similar to a related series of heterometallic manganese-dioxido clusters. The redox potentials of the [SrMn3O4] and [CaMn3O4] clusters are close, which is consistent with the observation that the OEC is functional only with one of these two metals. Considering our previous studies of [MMn3O2] moieties, the present results with more structurally accurate models of the OEC ([MMn3O4]) suggest a general relationship between the reduction potentials of heterometallic oxido clusters and the Lewis acidities of incorporated cations that applies to diverse structural motifs. These findings support proposals that one function of calcium in the OEC is to modulate the reduction potential of the cluster to allow electron transfer.

Mono- and bis-phosphine-ligated H93G myoglobin: spectral models for ferrous-phosphine and ferrous-CO cytochrome P450.

To further investigate the properties of phosphines as structural and functional probes of heme proteins, mono- and bis-phosphine [tris(hydroxymethyl)phosphine, THMP] adducts of H93G myoglobin (Mb) have been prepared by stepwise THMP titrations of exogenous ligand-free ferric and ferrous H93G Mb, respectively. Bubbling with CO or stepwise titration with imidazole (Im) of the bis-THMP-ligated ferrous protein generated a mixed ligand (THMP/CO or THMP/Im, respectively) ferrous complexes. Stable oxyferrous H93G(THMP) Mb was formed at -40°C by bubbling the mono-THMP-Fe(II) protein with O2. A THMP-ligated ferryl H93G Mb moiety has been partially formed upon addition of H2O2 to the ferric mono-THMP adduct. All the species prepared above have been characterized with UV-visible (UV-vis) absorption and magnetic circular dichroism (MCD) spectroscopy in this study. The six-coordinate ferrous bis-phosphine and mono-phosphine/CO complexes of H93G Mb exhibit characteristic spectral features (red-shifted Soret/unique-shaped MCD visible bands and hyperporphyrin spectra, respectively) that only have been seen for the analogous phosphine or CO-complexes of thiolate-ligated heme proteins such as cytochrome P450 (P450) and Caldariomyces fumago chloroperoxidase (CPO). However, such resemblance is not seen in phosphine-ligated ferric H93G Mb even though phosphine-bound ferric P450 and CPO display hyperporphyrin spectra. In fact, bis-THMP-bound ferric H93G Mb exhibits MCD and UV-vis absorption spectra that are similar to those of bis-amine- and bis-thioether-ligated H93G Mb complexes. This study also further demonstrates the utility of the H93G cavity mutant for preparing novel heme iron coordination structures.