Mechanistic Insights into the N-Hydroxylations Catalyzed by the Binuclear Iron Domain of SznF Enzyme: Key Piece in the Synthesis of Streptozotocin.

SznF, a member of the emerging family of heme-oxygenase-like (HO-like) di-iron oxidases and oxygenases, employs two distinct domains to catalyze the conversion of Nω-methyl-L-arginine (L-NMA) into N-nitroso-containing product, which can subsequently be transformed into streptozotocin. Using unrestricted density functional theory (UDFT) with the hybrid functional B3LYP, we have mechanistically investigated the two sequential hydroxylations of L-NMA catalyzed by SznF's binuclear iron central domain. Mechanism B primarily involves the O-O bond dissociation, forming Fe(IV)=O, induced by the H+/e- introduction to the FeA side of µ-1,2-peroxo-Fe2(III/III), the substrate hydrogen abstraction by Fe(IV)=O, and the hydroxyl rebound to the substrate N radical. The stochastic addition of H+/e- to the FeB side (mechanism C) can transition to mechanism B, thereby preventing enzyme deactivation. Two other competing mechanisms, involving the direct O-O bond dissociation (mechanism A) and the addition of H2O as a co-substrate (mechanism D), have been ruled out.

CMTM6 recruits T cells within the endocervical adenocarcinoma microenvironment and suppresses cell proliferation via the p53 pathway.

Endocervical adenocarcinoma (ECA), harboring poor prognosis, is divided into human papilloma virus (HPV)-associated adenocarcinoma (HPVA) and non-HPVA (NHPVA), each consisting of a heterogeneous immune microenvironment. We aim to examine the effect of CKLF-like MARVEL transmembrane domain 6 (CMTM6), a key regulator of PD-L1, on ECA. Immunohistochemistry and RNA-sequencing (RNA-seq) were used to detect CMTM6, Programmed death ligand 1 (PD-L1), and immune cells biomarkers levels in tumors. RT-qPCR and Western Blotting were used to detect the mRNA and protein level changed in cells. The expression of CMTM6 in ECA is upregulated compared to cervical squamous cell carcinoma tissues. More infiltrating T cells were observed in CMTM6high ECA tissues, especially in CMTM6high HPVA. Higher expression of CMTM6 is associated with a higher rate of infiltrating CD8+ T cells in HPVA, but not in NHPVA. ECA patients were divided into three groups according to the co-expression status of CMTM6 and PD-L1(CPS) . Patients with CMTM6high /PD-L1(CPS+) had the longest OS and DFS, especially in NHPVA patients. Moreover, knock down of CMTM6 promotes ECA cell proliferation via the p53 pathway. CMTM6 recruits T cells, suppresses ECA cell proliferation via the p53 pathway and can be used as a novel prognostic indicator for ECA patients.

Clinicopathologic features, tumor immune microenvironment and genomic landscape of Epstein-Barr virus-associated intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about Epstein-Barr virus (EBV)-associated intrahepatic cholangiocarcinoma (EBVaICC) because of its rarity. We aimed to comprehensively investigate the clinicopathology, tumor immune microenvironment (TIME) and genomic landscape of this entity in southern China. METHODS: We evaluated 303 intrahepatic cholangiocarcinomas (ICCs) using in situ hybridization for EBV. We compared clinicopathological parameters between EBVaICC and nonEBVaICC, and we analyzed EBV infection status, tumor-infiltrating lymphocytes (TILs) and genomic features of EBVaICC by immunohistochemistry, double staining, nested PCR, multiplex immunofluorescence staining, fluorescence in situ hybridization and whole-exome sequencing. RESULTS: EBVaICC accounted for 6.6% of ICCs and was associated with EBV latency type I infection and clonal EBV isolates. Patients with EBVaICC were more often female and younger, with solitary tumors, higher HBV infection rates and less frequent cirrhosis; the lymphoepithelioma-like (LEL) subtype was more common in EBVaICC. EBVaICC was associated with a significantly larger TIME component than nonEBVaICC. The LEL subtype of EBVaICC - associated with a significantly increased density and proportion of CD20+ B cells and CD8+ T cells - was associated with significantly higher 2-year survival rates than conventional EBVaICC and nonEBVaICC. Both PD-1 and PD-L1 in TILs, and PD-L1 in tumor cells, were overexpressed in EBVaICC. High PD-L1 expression in tumor cells and high CD8+ TIL densities were significantly more common in EBVaICC than in nonEBVaICC. Seven genes (MUC4, DNAH1, GLI2, LIPE, MYH7, RP11-766F14.2 and WDR36) were mutated in at least 3 patients. EBVaICC had a different mutational pattern to liver fluke-associated cholangiocarcinoma and HBV-associated ICC. CONCLUSIONS: EBVaICC, as a subset of ICC, has unique etiological, clinicopathological and genetic characteristics, with a significantly larger TIME component. Paradoxically, patients with EBVaICC could be candidates for immune checkpoint therapy. LAY SUMMARY: Epstein-Barr virus (EBV) is associated with a subtype of intrahepatic cholangiocarcinoma, with unique clinicopathological and genetic characteristics. The tumor immune microenvironment is also different in this tumor subtype and patients with EBV-associated intrahepatic cholangiocarcinoma may respond well to immune checkpoint inhibitors.

Radical 1,4-Aryl Migration Enabled Remote Cross-Electrophile Coupling of α-Amino-ß-Bromo Acid Esters with Aryl Bromides.

We report an unprecedented, efficient nickel-catalysed radical relay for the remote cross-electrophile coupling of ß-bromo-α-benzylamino acid esters with aryl bromides via 1,4-aryl migration/arylation cascades. ß-Bromo-α-benzylamino acid esters are considered as unique molecular scaffolds allowing for aryl migration reactions, which are conceptually novel variants for the radical Truce-Smiles rearrangement. This reaction enables the formation of two new C(sp3 )-C(sp2 ) bonds using a bench-stable Ni/bipyridine/Zn system featuring a broad substrate scope and excellent diastereoselectivity, which provides an effective platform for the remote aryl group migration and arylation of amino acid esters via redox-neutral C(sp3 )-C(sp2 ) bond cleavage. Mechanistically, this cascade reaction is accomplished by combining two powerful catalytic cycles consisting of a cross-electrophile coupling and radical 1,4-aryl migration through the generation of C(sp3 )-centred radical intermediates from the homolysis of C(sp3 )-Br bonds and the switching of the transient alkyl radical into a robust α-aminoalkyl radical.

Mechanism and Inhibitor Exploration with Binuclear Mg Ketol-Acid Reductoisomerase: Targeting the Biosynthetic Pathway of Branched-Chain Amino Acids.

Binuclear Mg ketol-acid reductoisomerase (KARI), which converts (S)-2-acetolactate into (R)-2,3-dihydroxyisovalerate, is responsible for the second step of the biosynthesis of branched-chain amino acids in plants and microorganisms and thus serves as a key inhibition target potentially without effects on mammals. Here, through the use of density functional calculations and a chemical model, the KARI-catalyzed reaction has been demonstrated to include the initial deprotonation of the substrate C2 hydroxy group, bridged by the two Mg ions, alkyl migration from the C2-alkoxide carbon atom to the C3-carbonyl carbon atom, and hydride transfer from a nicotinamide adenine dinucleotide phosphate [NAD(P)H] cofactor to C2. A dead-end mechanism with a hydride transferred to the C3 carbonyl group has been ruled out. The nucleophilicity (migratory aptitude) of the migrating carbon atom and the provision of additional negative charge to the di-Mg coordination sphere have significant effects on the steps of alkyl migration and hydride transfer, respectively. Other important mechanistic characteristics are also revealed. Inspired by the mechanism, an inhibitor (2-carboxylate-lactic acid) was designed and predicted by barrier analysis to be effective in inactivating KARI, hence probably enriching the antifungal and antibacterial library. Two types of slow substrate analogues (2-trihalomethyl acetolactic acids and 2-glutaryl lactic acid) were also found.

A Coiled-Coil Domain Containing 50 Splice Variant Is Modulated by Serine/Arginine-Rich Splicing Factor 3 and Promotes Hepatocellular Carcinoma in Mice by the Ras Signaling Pathway.

Deregulation of alternative splicing contributes to the malignant progression of cancer. Little is known about the significant alternative splicing events in hepatocellular carcinoma (HCC). High-throughput sequencing revealed that coiled-coil domain containing 50 (CCDC50) pre-mRNA is aberrantly spliced in 50% of our HCC cases. A BaseScope assay was performed to examine the expression of CCDC50S (a truncated oncogenic splice variant) in HCC tissues. Compared with benign liver tumors and several other types of solid tumors, CCDC50S mRNA was up-regulated in HCC, with a diagnostic potential (sensitivity, 0.711; specificity, 0.793). High expression of CCDC50S mRNA in HCC was significantly correlated with poor tumor differentiation, advanced tumor node metastasis (TNM) stage, and unfavorable prognosis. Overexpression of CCDC50S exerted tumorigenic activities that promoted HCC growth and metastasis by activation of Ras/forkhead box protein O4 (Foxo4) signaling. Either suppression of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation or overexpression of Foxo4 markedly attenuated CCDC50S-mediated phenotypes. Furthermore, serine- and arginine-rich splicing factor 3 (SRSF3) directly bound to CCDC50S mRNA to maintain its stability in the cytoplasm. The cytosolic retention of SRSF3 was mediated by the interaction of hepatitis B virus-encoded X protein (HBx) and 14-3-3ß. Ectopic HBx expression induced expression of cytosolic SRSF3 and CCDC50S. Conclusion: Our study provided compelling evidence that up-regulation of CCDC50S was modulated by HBx/SRSF3/14-3-3ß complex and enhanced oncogenic progression of HCC through the Ras/Foxo4 signaling pathway. These data suggest that CCDC50S may serve as a diagnostic and prognostic biomarker and probably a promising therapeutic target in HCC.

How To Produce Methane Precursor in the Upper Ocean by An Untypical Non-Heme Fe-Dependent Methylphosphonate Synthase?

A new methane formation pathway, which uses methylphosphonate (MPn) as the methane precursor, has been discovered in the upper ocean. Methylphosphonate synthase (MPnS) is a key piece in this pathway to produce MPn from 2-hydroxyethylphosphonate (2-HEP), using an untypical 2-His-1-Gln non-heme iron architecture. Herein, the MPnS reaction mechanism was demonstrated by the density functional calculations to mainly include the substrate hydroxyl deprotonation, the formation of a MPn radical and a formate, and the hydrogen abstraction of formate by MPn radical. The second-shell Lys28' may serve as a proton reservoir activating 2-HEP and regenerating the Fe site. The Fe-bound superoxide radical is a bifunctional species to deprotonate the substrate hydroxyl and abstract the substrate methylene hydrogen. Several alternative mechanisms have been ruled out. Furthermore, the catalytic activity of MPnS was found to be inactivated/reduced by the mutation of Gln152E/Gln152H/Gln152D, rendering a significant evolutionary advantage with an uncommon 2-His-1-Gln triad introduced to the ferrous coordination sphere.

Insights into the Chemical Reactivity in Acetyl-CoA Synthase.

The biological synthesis of acetyl-coenzyme A (acetyl-CoA), catalyzed by acetyl-CoA synthase (ACS), is of biological significance and chemical interest acting as a source of energy and carbon. The catalyst contains an unusual hexa-metal cluster with two nickel ions and a [Fe4S4] cluster. DFT calculations have been performed to investigate the ACS reaction mechanism starting from three different oxidation states (+2, +1, and 0) of Nip, the nickel proximal to [Fe4S4]. The results indicate that the ACS reaction proceeds first through a methyl radical transfer from cobalamin (Cbl) to Nip randomly accompanying with the CO binding. After that, C-C bond formation occurs between the Nip-bound methyl and CO, forming Nip-acetyl. The substrate CoA-S- then binds to Nip, allowing C-S bond formation between the Nip-bound acetyl and CoA-S-. Methyl transfer is rate-limiting with a barrier of ∼14 kcal/mol, which does not depend on the presence or absence of CO. Both the Nip2+ and Nip1+ states are chemically capable of catalyzing the ACS reaction independent of the state (+2 or +1) of the [Fe4S4] cluster. The [Fe4S4] cluster is not found to affect the steps of methyl transfer and C-C bond formation but may be involved in the C-S bond formation depending on the detailed mechanism chosen. An ACS active site containing a Nip(0) state could not be obtained. Optimizations always led to a Nip1+ state coupled with [Fe4S4]1+. The calculations show a comparable activity for Nip1+/[Fe4S4]1+, Nip1+/[Fe4S4]2+, and Nip2+/[Fe4S4]2+. The results here give significant insights into the chemistry of the important ACS reaction.

Asymmetric abstraction of two chemically-equivalent methylene hydrogens: significant enantioselectivity of endoperoxide presented by fumitremorgin B endoperoxidase.

The combination of the inert C-H bond activation and asymmetric synthesis, especially the transformation of prochiral sp3 precursors to chiral sp3 centers, is a profound challenge. In the present DFT calculations, the unique enantioselectivity in verruculogen biosynthesis catalyzed by fumitremorgin B endoperoxidase (FtmOx1) has been mechanistically investigated, where a prochiral methylene in fumitremorgin B is dominantly converted to an R-chiral eight-membered endoperoxy ring. FtmOx1 is the first-reported mononuclear α-ketoglutarate-dependent non-heme iron enzyme responsible for chiral endoperoxide formation, which handles the substrate using a Tyr224 radical resulting from the hydrogen abstraction by an FeIV[double bond, length as m-dash]O species. It is demonstrated that the perfect enantioselectivity of the R-endoperoxy ring originates from the asymmetric abstraction of two chemically-equivalent methylene hydrogens from substrate chain A by the Tyr224 radical and the high conformation stability of the resultant chain A radical due to steric effects. The barrier difference in the abstraction of two hydrogens is 5.6 kcal mol-1. The hydrogen abstraction by the Tyr224 radical is rate-limiting in the FtmOx1 reaction with an overall barrier of 18.6 kcal mol-1. The results obtained here advance the understanding of the chemistry in enantioselectivity, providing a potentially general way for the transformation of prochiral sp3 precursors to chiral sp3 centers.

Exothermic or Endothermic Decomposition of Disubstituted Tetrazoles Tuned by Substitution Fashion and Substituents.

Nitrogen-rich compounds such as tetrazoles are widely used as candidates in gas-generating agents. However, the details of the differentiation of the two isomers of disubstituted tetrazoles are rarely studied, which is very important information for designing advanced materials based on tetrazoles. In this article, pairs of 2,5- and 1,5-disubstituted tetrazoles were carefully designed and prepared for study on their thermal decomposition behavior. Also, the substitution fashion of 2,5- and 1,5- and the substituents at C-5 position were found to affect the endothermic or exothermic properties. This is for the first time to the best of our knowledge that the thermal decomposition properties of different tetrazoles could be tuned by substitution ways and substitute groups, which could be used as a useful platform to design advanced materials for temperature-dependent rockets. The aza-Claisen rearrangement was proposed to understand the endothermic decomposition behavior.

An Iron-Containing Metal-Organic Framework as a Highly Efficient Catalyst for Ozone Decomposition.

We present an iron-containing metal-organic framework, MIL-100(Fe), for ozone removal. MIL-100(Fe) exhibits long-lasting ozone conversion efficiency of 100 % for over 100 h under a relative humidity of 45 % and space velocity of 1.9×105  h-1 at room temperature, which is well beyond the performance of most porous or metal catalysts such as activated carbon and α-MnO2 . We also investigated the impact of humidity level and elucidated the plausible reaction mechanism, which is further confirmed by DFT calculations. Furthermore, MIL-100(Fe) can be processed into films and used as filtration layer in a mask to protect personnel against ozone contamination. This study demonstrates the promising potential of MOFs in ozone pollution control, and also offers new insights for the design of ozone decomposition catalysts.

From NAD+ to Nickel Pincer Complex: A Significant Cofactor Evolution Presented by Lactate Racemase.

Lactate racemase (LarA), a new nickel enzyme discovered recently, catalyzes the racemization between d- and l-lactates with a novel nickel pincer cofactor (Ni-PTTMN) derived from nicotinic acid. In this study, by using DFT and a 200-atom active-site model, LarA is revealed to employ a modified proton-coupled hydride-transfer mechanism in which a hydride is transferred to a cofactor pyridine carbon from the substrate α-carbon along with proton transfer from the substrate hydroxy group to a histidine, and then moved back from the opposite side. Tyr294 and Lys298 provide significant acceleration effects by orientating substrates and stabilizing the negative charge developing at the substrate hydroxy oxygen. The barrier was determined to be 12.0 kcal mol-1 , which reveals enhanced racemase activity relative to the LarA reaction using NAD+ -like cofactors. Compared with NAD+ , Ni-PTTMN has a stronger hydride-addition reactivity in moderate and high environmental polarity and may fit perfectly the moderately polar active site of LarA.

Unraveling the Mechanism and Regioselectivity of the B12-Dependent Reductive Dehalogenase PceA.

PceA is a cobalamin-dependent reductive dehalogenase that catalyzes the dechlorination of perchloroethylene to trichloroethylene and then to cis-dichloroethylene as the sole final product. The reaction mechanism and the regioselectivity of this enzyme are investigated by using density functional calculations. Four different substrates, namely, perchloroethylene, trichloroethylene, cis-dichloroethylene, and chlorotheylene, have been considered and were found to follow the same reaction mechanism pattern. The reaction starts with the reduction of Co(II) to Co(I) through a proton-coupled electron transfer process, with the proton delivered to a Tyr246 anion. This is followed by concerted C-Cl bond heterolytic cleavage and proton transfer from Tyr246 to the substrate carbon atom, generating a Co(III) -Cl intermediate. Subsequently, a one-electron transfer leads to the formation of the Co(II) -Cl product, from which the chloride and the dehalogenated product can be released from the active site. The substrate reactivity follows the trend perchloroethylene>trichloroethylene≫cis-dichloroethylene≫chlorotheylene. The barriers for the latter two substrates are significantly higher compared with those for perchloroethylene and trichloroethylene, implying that PceA does not catalyze their degradation. In addition, the formation of cis-dichloroethylene has a lower barrier by 3.8 kcal mol(-1) than the formation of trans-dichloroethylene and 1,1-dichloroethylene, reproducing the regioselectivity. These results agree quite well with the experimental findings, which show cis-dichloroethylene as the sole product in the PceA-catalyzed dechlorination of perchloethylene and trichloroethylene.

The Decarboxylation of α,ß-Unsaturated Acid Catalyzed by Prenylated FMN-Dependent Ferulic Acid Decarboxylase and the Enzyme Inhibition.

Ferulic acid decarboxylase (Fdc1) is able to catalyze the decarboxylation of α,ß-unsaturated acids using a novel cofactor, prenylated flavin mononucleotide (PrFMN). Using density functional theory calculations, we here have investigated the Fdc1 reaction mechanism with the substrate of α-methylcinnamic acid. It is demonstrated that Fdc1 employs a 1,3-dipolar cycloaddition mechanism involving four concerted steps, where the Glu282 acts as a crucial proton donor to protonate the α carbon (Cα). The last step, the decomposition of a pyrrolidine species, is rate-limiting with an overall barrier of 18.9 kcal mol-1. Furthermore, when α-hydroxycinnamic acid is used, the Glu282 is found to have another face to transport the hydroxyl proton to the Cß atom to promote the tautomerization from enol intermediate to ketone species leading to the inhibition of the Fdc1 enzyme. The PrFMN roles are also discussed in detail.

A promising candidate with D-A-A-A architecture as an efficient sensitizer for dye-sensitized solar cells.

A series of metal-free organic dyes with electron-rich (D) and electron-deficient units (A) as π linkers have been studied theoretically by means of density functional theory (DFT) and time-dependent DFT calculations to explore the effects of π spacers on the optical and electronic properties of triphenylamine dyes. The results show that Dye 1 with a structure of D-A-A-A is superior to the typical C218 dye in various key aspects, including the maximum absorption (λmax =511 nm), the charge-transfer characteristics (D/Δq/t is 5.49 Å/0.818 e(-) /4.41 Å), the driving force for charge-carrier injection (ΔGinject =1.35 eV)/dye regeneration (ΔGregen =0.27 eV), and the lifetime of the first excited state (τ=3.1 ns). It is thus proposed to be a promising candidate in dye-sensitized solar cell applications.

Phosphate monoester hydrolysis by trinuclear alkaline phosphatase; DFT study of transition States and reaction mechanism.

Alkaline phosphatase (AP) is a trinuclear metalloenzyme that catalyzes the hydrolysis of a broad range of phosphate monoesters to form inorganic phosphate and alcohol (or phenol). In this paper, by using density functional theory with a model based on a crystal structure, the AP-catalyzed hydrolysis of phosphate monoesters is investigated by calculating two substrates, that is, methyl and p-nitrophenyl phosphates, which represent alkyl and aryl phosphates, respectively. The calculations confirm that the AP reaction employs a "ping-pong" mechanism involving two chemical displacement steps, that is, the displacement of the substrate leaving group by a Ser102 alkoxide and the hydrolysis of the phosphoseryl intermediate by a Zn2-bound hydroxide. Both displacement steps proceed via a concerted associative pathway no matter which substrate is used. Other mechanistic aspects are also studied. Comparison of our calculations with linear free energy relationships experiments shows good agreement.

Iodinated Al(III)-based phthalocyanines are promising sensitizers for dye-sensitized solar cells; a theoretical comparison between Zn(II), Mg(II), and Al(III)-based phthalocyanine sensitizers.

To design efficient dyes for dye-sensitized solar cells (DSSCs), using a Zn-coordinated phthalocyanine (TT7) as the prototype, a series of phthalocyanine dyes (Pcs) with different metal ions and peripheral/axial groups have been investigated by means of density functional theory (DFT) and time-dependent DFT (TDDFT) methods. Computational results show that the iodinated Al-based dye with a peripheral amino group (Al-I-NH2-Pc) exhibits the largest redshift in the maximum absorbance (λ(max)). In addition, Al-based dyes have appropriate energy-level arrangements of frontier orbitals to keep excellent balance between electron injection and regeneration of oxidized dyes. Further, it has been found that the intermolecular π-staking interaction in Al-I-Pc molecules is weaker than the other metal-based Pcs, which may effectively reduce dye aggregation on the semi-conductor surface. All these results suggest iodinated Al-based Pcs (Al-I-Pcs) to be potentially promising sensitizers in DSSCs.

Symmetry breaking of α-[H2W12O40](6-) depends on the transformation of isopolyoxotungstates.

Two enantiotopic 1D chain compounds, [Cu3(L1)3(H2O)2(H2W12O40)]·4H2O (1a,b; L1 = 2-(4,6-bis(pyridin-2-yl)pyridin-2-yl)pyridine), crystallizing in the chiral space group P212121 were prepared and spontaneously resolved in the absence of any chiral source. Interestingly, compounds 1a,b can be prepared from a [W7O24](6-) aqueous solution, [(n-C4H9)4N]4[W10O32], or Na10[H2W12O42], but when [H2W12O40](6-) aqueous solution was the starting material, the achiral compound [CuL1]2[H4W12O40]·5H2O (2) was obtained. When a terpyridine ligand (L2) having a coordination mode similar to that of L1 was used, the mesomeric dimer [Cu3(L2)3(H2O)(H2W12O40)]2·4H2O (3) was obtained from [W7O24](6-) aqueous solution or Na10[H2W12O42], but from [H2W12O40](6-) aqueous solution only compound [Cu2(L2)2Cl2]2[W10O32] (4) was isolated. It is notable that in compounds 1a,b and 3 the symmetry of the α-[H2W12O40](6-) cluster is broken by asymmetric coordination with metal-organic units in a similar mode. As the asymmetric subunit based on a tridecorated [H2W12O40](6-) cluster can be obtained from several isopolyoxotungstate sources except for [H2W12O40](6-), we speculate that the symmetry breaking of α-[H2W12O40](6-) depends on the transformation of isopolyoxotungstates. Furthermore, during the transformation a possible reaction intermediate as the precursor for 1a,b, compound [Cu3(L1)3(H2O)3(H4W11O38)] (5), has been presented and characterized by density functional theory (DFT) calculations.

An investigation of possible competing mechanisms for Ni-containing methyl-coenzyme M reductase.

Ni-containing methyl-coenzyme M reductase (MCR) is capable of catalyzing methane formation from methyl-coenzyme M (CH3-SCoM) and coenzyme B (CoB-SH), and also its reverse reaction (methane oxidation). Based on extensive experimental and theoretical investigations, it has turned out that a mechanism including an organometallic methyl-Ni(III)F430 intermediate is inaccessible, while another mechanism involving a methyl radical and a Ni(II)-SCoM species currently appears to be the most acceptable one for MCR. In the present paper, using hybrid density functional theory and an active-site model based on the X-ray crystal structure, two other mechanisms were studied and finally also ruled out. One of them, involving proton binding on the CH3-SCoM substrate, which should facilitate methyl-Ni(III)F430 formation, is demonstrated to be quite unfavorable since the substrate has a much smaller proton affinity than the F430 cofactor. Another one (oxidative addition mechanism) is also shown to be unfavorable for the MCR reaction, due to the large endothermicity for the formation of the ternary intermediate with side-on C-S (for CH3-SCoM) or C-H (for methane) coordination to Ni.

How is methane formed and oxidized reversibly when catalyzed by Ni-containing methyl-coenzyme M reductase?

Ni-containing methyl-coenzyme M reductase (MCR) is capable of catalyzing methane formation and has recently been observed to also be able to catalyze the reverse reaction, the anaerobic oxidation of methane. The forward reaction has been extensively studied theoretically before and was found to consist of two steps. The first step is rate-limiting and the second step was therefore treated at a lower level. For an accurate treatment of the reverse reaction, both steps have to be studied at the same level. In the present paper, the mechanisms for the reversible formation and oxidation of methane catalyzed by MCR have been investigated using hybrid density functional theory with recent developments, in particular including dispersion effects. An active-site model was constructed based on the X-ray crystal structure. The calculations indicate that the MCR reaction is indeed reversible and proceeds via a methyl radical and a Ni-S(CoM) intermediate with reasonable reaction barriers in both directions. In a competing mechanism, the formation of the crucial Ni-methyl intermediate, was found to be strongly endergonic by over 20 kcal mol(-1) (including a barrier) with dispersion and entropy effects considered, and thus would not be reachable in a reasonable time under natural conditions.