RESUMEN
Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (â¼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.
Asunto(s)
Cobre , Nitrito Reductasas , Nitritos , Catálisis , Cobre/química , Nitrito Reductasas/química , Nitritos/química , Oxidación-Reducción , Análisis EspectralRESUMEN
The link of an antenna dye with an electron spin converter, in this case naphthalenediimide and C60, produces a system with a rich photophysics including the detection of more than one triplet state on the long timescale (tens of µs). Beside the use of optical spectroscopies in the ns and in the fs time scale, we used time-resolved Electron Paramagnetic Resonance (TREPR) to study the system evolution following photoexcitation. TREPR keeps track of the formation path of the triplet states through specific spin polarization patterns observed in the spectra. The flexibility of the linker and solvent polarity play a role in favouring either electron transfer or energy transfer processes.
RESUMEN
We report a Cu-catalyzed oxidative coupling of aliphatic amines with benzylic and aliphatic boronic esters to give high value alkyl amines, products found widely in applications from medicinal chemistry to materials science. This operationally simple reaction, which can be performed on gram scale, runs under mild conditions and exhibits broad functional group tolerance. The terminal oxidant of the reaction is O2 from the air, avoiding the need for additional chemical oxidants. Investigation into the reaction mechanism suggests that the boronic ester is activated by an aminyl radical, formed through oxidation of the amine by the Cu catalyst, to give a key alkyl radical intermediate. To demonstrate its utility and potential for late-stage functionalization, we showcase the method as the final step in the total synthesis of a TRPV1 antagonist.
RESUMEN
Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed "methionine track" leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Cobre/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Iones , Masculino , Metionina/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismoRESUMEN
Polymeric frustrated Lewis pairs, or poly(FLP)s, have served to bridge the gap between functional polymer science and main group catalysis, pairing the uniqueness of sterically frustrated Lewis acids and bases with a polymer scaffold to create self-healing gels and recyclable catalysts. However, their utilization in radical chemistry is unprecedented. In this paper, we disclose the synthesis of polymeric frustrated radical pairs, or poly(FRP)s, by in situ photoinduction of FLP moieties, where their Lewis acidic and basic centers are tuned to promote single electron transfer (SET). Through systematic manipulation of the chemical structure, we demonstrate that inclusion of ortho-methyl groups on phosphine monomers is crucial to enable SET. The generation of radicals is evidenced by monitoring the stable polymeric phosphine radical cations via UV/vis and EPR spectroscopy. These new poly(FRP)s enable both catalytic hydrogenation and radical-mediated photocatalytic perfluoroalkylations. These polymeric radical systems open new avenues to design novel functional polymers for catalysis and photoelectrical chemistry.
RESUMEN
Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
Asunto(s)
Cobre , Histidina , Oxigenasas de Función Mixta , Estrés Oxidativo , CatálisisRESUMEN
'Bacterial-type' ferredoxins host a cubane [4Fe4S]2+/+ cluster that enables these proteins to mediate electron transfer and facilitate a broad range of biological processes. Peptide maquettes based on the conserved cluster-forming motif have previously been reported and used to model the ferredoxins. Herein we explore the integration of a [4Fe4S]-peptide maquette into a H2 -powered electron transport chain. While routinely formed under anaerobic conditions, we illustrate by electron paramagnetic resonance (EPR) analysis that these maquettes can be reconstituted under aerobic conditions by using photoactivated NADH to reduce the cluster at 240â K. Attempts to tune the redox properties of the iron-sulfur cluster by introducing an Fe-coordinating selenocysteine residue were also explored. To demonstrate the integration of these artificial metalloproteins into a semi-synthetic electron transport chain, we utilize a ferredoxin-inspired [4Fe4S]-peptide maquette as the redox partner in the hydrogenase-mediated oxidation of H2 .
Asunto(s)
Hidrogenasas , Proteínas Hierro-Azufre , Ferredoxinas/metabolismo , Proteínas Hierro-Azufre/química , Hidrogenasas/metabolismo , Oxidación-Reducción , Péptidos/metabolismo , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron-electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures.
Asunto(s)
Calmodulina , Simulación de Dinámica Molecular , Calcio/metabolismo , Calmodulina/química , Electrones , Unión Proteica , Conformación Proteica , Marcadores de SpinRESUMEN
Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 Å apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.
Asunto(s)
Carnitina/metabolismo , Oxigenasas de Función Mixta/metabolismo , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/química , Conformación Proteica , Especificidad por SustratoRESUMEN
Chemical recycling is one of the most promising technologies that could contribute to circular economy targets by providing solutions to plastic waste; however, it is still at an early stage of development. In this work, we describe the first light-driven, acid-catalyzed protocol for chemical recycling of polystyrene waste to valuable chemicals under 1 bar of O2. Requiring no photosensitizers and only mild reaction conditions, the protocol is operationally simple and has also been demonstrated in a flow system. Electron paramagnetic resonance (EPR) investigations and density functional theory (DFT) calculations indicate that singlet oxygen is involved as the reactive oxygen species in this degradation process, which abstracts a hydrogen atom from a tertiary C-H bond, leading to hydroperoxidation and subsequent C-C bond cracking events via a radical process. Notably, our study indicates that an adduct of polystyrene and an acid catalyst might be formed in situ, which could act as a photosensitizer to initiate the formation of singlet oxygen. In addition, the oxidized polystyrene polymer may play a role in the production of singlet oxygen under light.
Asunto(s)
Poliestirenos , Oxígeno Singlete , Catálisis , Luz , Oxidación-Reducción , Fármacos Fotosensibilizantes/química , Oxígeno Singlete/químicaRESUMEN
The oxidative cleavage of CâC double bonds with molecular oxygen to produce carbonyl compounds is an important transformation in chemical and pharmaceutical synthesis. In nature, enzymes containing the first-row transition metals, particularly heme and non-heme iron-dependent enzymes, readily activate O2 and oxidatively cleave CâC bonds with exquisite precision under ambient conditions. The reaction remains challenging for synthetic chemists, however. There are only a small number of known synthetic metal catalysts that allow for the oxidative cleavage of alkenes at an atmospheric pressure of O2, with very few known to catalyze the cleavage of nonactivated alkenes. In this work, we describe a light-driven, Mn-catalyzed protocol for the selective oxidation of alkenes to carbonyls under 1 atm of O2. For the first time, aromatic as well as various nonactivated aliphatic alkenes could be oxidized to afford ketones and aldehydes under clean, mild conditions with a first row, biorelevant metal catalyst. Moreover, the protocol shows a very good functional group tolerance. Mechanistic investigation suggests that Mn-oxo species, including an asymmetric, mixed-valent bis(µ-oxo)-Mn(III,IV) complex, are involved in the oxidation, and the solvent methanol participates in O2 activation that leads to the formation of the oxo species.
RESUMEN
Oxidation of quaternary ammonium substrate, carnitine by non-heme iron containing Acinetobacter baumannii (Ab) oxygenase CntA/reductase CntB is implicated in the onset of human cardiovascular disease. Herein, we develop a blue-light (365â nm) activation of NADH coupled to electron paramagnetic resonance (EPR) measurements to study electron transfer from the excited state of NADH to the oxidized, Rieske-type, [2Fe-2S]2+ cluster in the AbCntA oxygenase domain with and without the substrate, carnitine. Further electron transfer from one-electron reduced, Rieske-type [2Fe-2S]1+ center in AbCntA-WT to the mono-nuclear, non-heme iron center through the bridging glutamate E205 and subsequent catalysis occurs only in the presence of carnitine. The electron transfer process in the AbCntA-E205A mutant is severely affected, which likely accounts for the significant loss of catalytic activity in the AbCntA-E205A mutant. The NADH photo-activation coupled with EPR is broadly applicable to trap reactive intermediates at low temperature and creates a new method to characterize elusive intermediates in multiple redox-centre containing proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carnitina/metabolismo , Luz , Microbiota , Oxidorreductasas/metabolismo , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/aislamiento & purificación , Proteínas Bacterianas/genética , Carnitina/química , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Humanos , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Mutagénesis Sitio-Dirigida , NAD/química , Oxidación-Reducción , Oxidorreductasas/genéticaRESUMEN
Fatty acid photodecarboxylases (FAP) are a recently discovered family of FAD-containing, light-activated enzymes, which convert fatty acids to n-alkanes/alkenes with potential applications in the manufacture of fine and speciality chemicals and fuels. Poor catalytic stability of FAPs is however a major limitation. Here, we describe a methodology to purify catalytically stable and homogeneous samples of recombinant Chlorella variabilis NC64A FAP (CvFAP) from Escherichia coli. We demonstrate however that blue light-exposure, which is required for photodecarboxylase activity, also leads to irreversible inactivation of the enzyme, especially in the absence of palmitate substrate. Photoinactivation is attributed to formation of protein based organic radicals, which were observed by EPR spectroscopy. To suppress photoinactivation, we prepared stable and catalytically active FAP in the dark. The steady-state kinetic parameters of CvFAP (kcat: 0.31 ± 0.06 s-1 and KM: 98.8 ± 53.3 µM) for conversion of palmitic acid to pentadecane were determined using gas chromatography. Methods described here should now enable studies of the catalytic mechanism and exploitation of FAPs in biotechnology.
Asunto(s)
Carboxiliasas/metabolismo , Ácidos Grasos/metabolismo , Biocatálisis , Carboxiliasas/química , Escherichia coli/enzimología , Ácidos Grasos/química , Radicales Libres/química , Radicales Libres/metabolismo , Cinética , Procesos FotoquímicosRESUMEN
Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read-out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter-copper electron transfer catalyzed by CuNiRs. Cryoreduction-EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox-center containing proteins (homo and hetero metal ions).
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Nitrito Reductasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Oxidación-Reducción , TemperaturaRESUMEN
The reaction of [Ni(COD)2] (COD; cyclooctadiene) in THF with the NNN-pincer ligand bis(imino)pyridyl (L1) reveals a susceptibility to oxidation in an inert atmosphere ([O2] level <0.5 ppm), resulting in a transient Ni:dioxygen adduct. This reactive intermediate abstracts a hydrogen atom from THF and stabilizes an uncommon Ni(III) complex. The complex is crystallographically characterized by a molecular formula of [NiIII(L1··)2-(OH)] (1). Various isotopically labeled experiments (16O/18O) assertively endorse the origin of terminal oxygen based ligand in 1 due to the activation of molecular dioxygen. The presence of proton bound to the terminal oxygen in 1 is well supported by NMR, IR spectroscopy, DFT calculations, and hydrogen atom transfer (HAT) reactions promoted by 1. The observation of shakeup satellite peaks for the primary photoelectron lines of Ni(2p) in the X-ray photoelectron spectroscopy (XPS) unambiguously confirms the paramagnetic signature associated with the distorted square planar nickel ion, which is consistent with the trivalent oxidation state assigned for the nickel ion in 1. The variable temperature magnetic susceptibility data of 1 shows dominant antiferromagnetic interactions exist among the paramagnetic centers, resulting in an overall S = 1/2 ground state. Variable temperature X-band EPR studies performed on 1 show evidence for the S = 1/2 ground state, which is consistent with magnetic data. The unusual g-tensor extracted for the ground state S = 1/2 is analyzed under a strong exchange limit of spin-coupled centers. The electronic structure predicted for 1 is in good agreement with theoretical calculations.
RESUMEN
N-heterocyclic carbene (NHC) ligands have had a major impact in homogeneous catalysis, however, their potential role in biological systems is essentially unexplored. We replaced a copper-coordinating histidine (His) in the active site of the redox enzyme azurin with exogenous dimethyl imidazolylidene. This NHC rapidly restores the type-1 Cu center, with spectroscopic properties (EPR, UV/Vis) that are identical to those from N-coordination of the His in the wild type. However, the introduction of the NHC markedly alters the redox potential of the metal, which is a key functionality of this blue copper protein. These results suggest that C-bonding for histidine is plausible and a potentially relevant bonding mode of redox-active metalloenzymes in their (transient) active states.
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Azurina/química , Metano/análogos & derivados , Azurina/genética , Azurina/metabolismo , Dominio Catalítico , Cobre/química , Técnicas Electroquímicas , Espectroscopía de Resonancia por Spin del Electrón , Compuestos Heterocíclicos/química , Histidina/química , Ligandos , Metano/química , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , EspectrofotometríaRESUMEN
Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S â Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Cristalografía por Rayos X/métodos , Cisteína/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Hidrocarburo de Aril Hidroxilasas/química , Criopreservación , Cisteína/química , Familia 2 del Citocromo P450 , Enlace de HidrógenoRESUMEN
Activation of the diferrous center of the ß2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied (57)Fe, (14,15)N, (17)O, and (1)H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of (17)O and (1)H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH(-))Fe(III)-O-Fe(IV)]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the (14)N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238.
Asunto(s)
Escherichia coli/enzimología , Modelos Moleculares , Ribonucleótido Reductasas/química , Hierro/químicaRESUMEN
We report here an ENDOR study of an S = 1/2 intermediate state trapped during reduction of the binuclear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR spectra of this state confirm that the (63,65)Cu nuclei exhibits strong and almost entirely isotropic coupling to the unpaired electron, show that this coupling atypically has a positive sign, aiso = +148 MHz, and indicate an apparently undetectably small quadrupolar coupling. When the intermediate is generated using (13)CO, coupling to the (13)C is observed, with aiso = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase, in conjunction with complementary computational studies, leads us to conclude that the intermediate contains a partially reduced Mo(V)/Cu(I) center with CO bound at the copper. Our results provide strong experimental support for a reaction mechanism that proceeds from a comparable complex of CO with fully oxidized Mo(VI)/Cu(I) enzyme.
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Aldehído Oxidorreductasas/metabolismo , Alphaproteobacteria/enzimología , Cobre/metabolismo , Complejos Multienzimáticos/metabolismo , Aldehído Oxidorreductasas/química , Alphaproteobacteria/química , Alphaproteobacteria/metabolismo , Dominio Catalítico , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Complejos Multienzimáticos/químicaRESUMEN
Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a nonstoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.