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Polysaccharide monooxygenase (PMO) catalysis involves the chemically difficult hydroxylation of unactivated C-H bonds in carbohydrates. The reaction requires reducing equivalents and will utilize either oxygen or hydrogen peroxide as a cosubstrate. Two key mechanistic questions are addressed here: 1) How does the enzyme regulate the timely and tightly controlled electron delivery to the mononuclear copper active site, especially when bound substrate occludes the active site? and 2) How does this electron delivery differ when utilizing oxygen or hydrogen peroxide as a cosubstrate? Using a computational approach, potential paths of electron transfer (ET) to the active site copper ion were identified in a representative AA9 family PMO from Myceliophthora thermophila ( Mt PMO9E). When Y62, a buried residue 12 Å from the active site, is mutated to F, lower activity is observed with O 2 . However, a WT-level activity is observed with H 2 O 2 as a cosubstrate indicating an important role in ET for O 2 activation. To better understand the structural effects of mutations to Y62 and axial copper ligand Y168, crystal structures were solved of the wild type Mt PMO9E and the variants Y62W, Y62F, and Y168F. A bioinformatic analysis revealed that position 62 is conserved as either Y or W in the AA9 family. The Mt PMO9E Y62W variant has restored activity with O 2 . Overall, the use of redox-active residues to supply electrons for the reaction with O 2 appears to be widespread in the AA9 family. Furthermore, the results provide a molecular framework to understand catalysis with O 2 versus H 2 O 2 ., Competing Interests: Competing interests statement:The authors declare no competing interest.

Fungal polysaccharide monooxygenases (PMOs) oxidatively degrade cellulose and other carbohydrate polymers via a mononuclear copper active site using either O 2 or H 2 O 2 as a cosubstrate. Cellulose-active fungal PMOs in the auxiliary activity 9 (AA9) family have a conserved second-sphere hydrogen-bonding network consisting of histidine, glutamine, and tyrosine residues. The second-sphere histidine has been hypothesized to play a role in proton transfer in the O 2 -dependent PMO reaction. Here the role of the second-sphere histidine (H157) in an AA9 PMO, Mt PMO9E, was investigated. This PMO is active on soluble cello-oligosaccharides such as cellohexaose (Glc6), thus enabling kinetic analysis with the point variants H157A and H157Q. The variants appeared to fold similarly to the wild-type (WT) enzyme and yet exhibited weaker affinity toward Glc6 than WT (WT K D = 20 ± 3 μM). The variants had comparable oxidase (O 2 reduction to H 2 O 2 ) activity to WT at all pH values tested. Using O 2 as a cosubstrate, the variants were less active for Glc6 hydroxylation than WT, with H157A being the least active. Similarly, H157Q was competent for Glc6 hydroxylation with H 2 O 2 , but H157A was less active. Comparison of the crystal structures of H157Q and WT Mt PMO9E reveals that a terminal heteroatom of Q157 overlays with N ε of H157. Altogether, the data suggest that H157 is not important for proton transfer, but support a role for H157 as a hydrogen-bond donor to diatomic-oxygen intermediates, thus facilitating catalysis with either O 2 or H 2 O 2 .

Electrocatalytic water oxidation is a key transformation in many strategies designed to harness solar energy and store it as chemical fuels. Understanding the mechanism(s) of the best electrocatalysts for water oxidation has been a fundamental chemical challenge for decades. Here, we quantitate evolved dioxygen isotopologue composition via gas-phase EPR spectroscopy to elucidate the mechanisms of water oxidation on metal oxide electrocatalysts with high precision. Isotope fractionation is paired with computational and kinetic modeling, showing that this technique is sensitive enough to differentiate O-O bond-forming steps. Strong agreement between experiment and theory indicates that for the nickel-iron layered double hydroxide─one of the best earth-abundant electrocatalysts to be studied─water oxidation proceeds via a dioxo coupling mechanism to form a side-bound peroxide rather than a hydroxide attack to form an end-bound peroxide.

Blast disease in cereal plants is caused by the fungus Magnaporthe oryzae and accounts for a significant loss in food crops. At the outset of infection, expression of a putative polysaccharide monooxygenase ( Mo PMO9A) is increased. Mo PMO9A contains a catalytic domain predicted to act on cellulose and a carbohydrate-binding domain that binds chitin. A sequence similarity network of the Mo PMO9A family AA9 showed that 220 of the 223 sequences in the Mo PMO9A-containing cluster of sequences have a conserved unannotated region with no assigned function. Expression and purification of the full length and two Mo PMO9A truncations, one containing the catalytic domain and the domain of unknown function (DUF) and one with only the catalytic domain, were carried out. In contrast to other AA9 polysaccharide monooxygenases (PMOs), Mo PMO9A is not active on cellulose but showed activity on cereal-derived m ixed (1→3, 1→4)- β -D- g lucans (MBG). Moreover, the DUF is required for activity. Mo PMO9A exhibits activity consistent with C4 oxidation of the polysaccharide and can utilize either oxygen or hydrogen peroxide as a cosubstrate. It contains a predicted 3-dimensional fold characteristic of other PMOs. The DUF is predicted to form a coiled-coil with six absolutely conserved cysteines acting as a zipper between the two α-helices. Mo PMO9A substrate specificity and domain architecture are different from previously characterized AA9 PMOs. The results, including a gene ontology analysis, support a role for Mo PMO9A in MBG degradation during plant infection. Consistent with this analysis, deletion of Mo PMO9A results in reduced pathogenicity.

Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr (cell wall remodeling) , encodes two linked genes, cwr-1 and cwr-2 . Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the catalytic (PMO) domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein-protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR-1 PMO domain., Competing Interests: TD, AR, RS, AG, MM, NG No competing interests declared, (© 2022, Detomasi, Rico-Ramírez et al.)

Ligand-based mixed valent (MV) complexes of Al(iii) incorporating electron donating (ED) and electron withdrawing (EW) substituents on bis(imino)pyridine ligands (I 2 P) have been prepared. The MV states containing EW groups are both assigned as Class II/III, and those with ED functional groups are Class III and Class II/III in the (I 2 P - )(I 2 P 2- )Al and [(I 2 P 2- )(I 2 P 3- )Al] 2- charge states, respectively. No abrupt changes in delocalization are observed with ED and EW groups and from this we infer that ligand and metal valence p-orbitals are well-matched in energy and the absence of LMCT and MLCT bands supports the delocalized electronic structures. The MV ligand charge states (I 2 P - )(I 2 P 2- )Al and [(I 2 P 2- )(I 2 P 3- )Al] 2- show intervalence charge transfer (IVCT) transitions in the regions 6850-7740 and 7410-9780 cm -1 , respectively. Alkali metal cations in solution had no effect on the IVCT bands of [(I 2 P 2- )(I 2 P 3- )Al] 2- complexes containing -PhNMe 2 or -PhF 5 substituents. Minor localization of charge in [(I 2 P 2- )(I 2 P 3- )Al] 2- was observed when -PhOMe substituents are included., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Iron-doped nickel layered double hydroxides (LDHs) are among the most active heterogeneous water oxidation catalysts. Due to interspin interactions, however, the high density of magnetic centers results in line-broadening in magnetic resonance spectra. As a result, gaining atomic-level insight into the catalytic mechanism via electron paramagnetic resonance (EPR) is not generally possible. To circumvent spin-spin broadening, iron and nickel atoms were doped into nonmagnetic [ZnAl]-LDH materials and the coordination environments of the isolated Fe(III) and Ni(II) sites were characterized. Multifrequency EPR spectroscopy identified two distinct Fe(III) sites ( S = 5/2) in [Fe:ZnAl]-LDH. Changes in zero field splitting (ZFS) were induced by dehydration of the material, revealing that one of the Fe(III) sites was solvent-exposed (i.e., at an edge, corner, or defect site). These solvent-exposed sites featured an axial ZFS of 0.21 cm -1 when hydrated. The ZFS increased dramatically upon dehydration (to -1.5 cm -1 ), owing to lower symmetry and a decrease in the coordination number of iron. The ZFS of the other ("inert") Fe(III) site maintained an axial ZFS of 0.19-0.20 cm -1 under both hydrated and dehydrated conditions. We observed a similar effect in [Ni:ZnAl]-LDH materials; notably, Ni(II) ( S = 1) atoms displayed a single, small ZFS (±0.30 cm -1 ) in hydrated material, whereas two distinct Ni(II) ZFS values (±0.30 and ±1.1 cm -1 ) were observed in the dehydrated samples. Although the magnetically dilute materials were not active catalysts, the identification of model sites in which the coordination environments of iron and nickel were particularly labile (e.g., by simple vacuum drying) is an important step toward identifying sites in which the coordination number may drop spontaneously in water, a probable mechanism of water oxidation in functional materials.

S -Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S -adenosyl methionine to generate the 5'-deoxyadenosyl radical (5'dAdo • ), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5'dAdo • radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe-S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO) 2 CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe-Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the C α H-NH 2 with a CH 2 at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and 13 C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C 2 =C 3 alkene substitution relative to HPPA's single bond. Interestingly, the HydG reaction with the cis-p -coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p -coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5'dAdo • radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p -coumaric acid. Density functional theory energetics calculations show that the cis-p -coumaric acid has approximately the same C-H bond dissociation free energy as 5'dAdo • , providing a possible explanation for our ability to trap an appreciable fraction of 5'dAdo • in this specific rSAM reaction. The radical's EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5'dAdo • radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5'dAdo • radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Water-stable organic mixed valence (MV) compounds have been prepared by the reaction of reduced bis(imino)pyridine ligands (I 2 P) with the trichloride salts of Al, Ga, and In. The coordination of two tridentate ligands to each ion affords octahedral complexes that are accessible with five ligand charge states: [(I 2 P 0 )(I 2 P - )M] 2+ , [(I 2 P - ) 2 M] + , (I 2 P - )(I 2 P 2- )M, [(I 2 P 2- ) 2 M] - , and [(I 2 P 2- )(I 2 P 3- )M] 2- , and for M = Al only, [(I 2 P 3- ) 2 M] 3- . In solid-state structures, the anionic members of the redox series are stabilized by the intercalation of Na + cations within the ligands. The MV members of the redox series, (I 2 P - )(I 2 P 2- )M and [(I 2 P 2- )(I 2 P 3- )M] 2- , show characteristic intervalence transitions, in the near-infrared regions between 6800-7400 and 7800-9000 cm -1 , respectively. Cyclic voltammetry (CV), NIR spectroscopic, and X-ray structural studies support the assignment of class II for compounds [(I 2 P 2- )(I 2 P 3- )M] 2- and class III for M = Al and Ga in (I 2 P - )(I 2 P 2- )M. All compounds containing a singly reduced I 2 P - ligand exhibit a sharp, low-energy transition in the 5100-5600 cm -1 region that corresponds to a π*-π* transition. CV studies demonstrate that the electron-transfer events in each of the redox series, Al, Ga, and In, span 2.2, 1.4, and 1.2 V, respectively.

Electrochemical generation of ammonia (NH 3 ) from nitrogen (N 2 ) using renewable electricity is a desirable alternative to current NH 3 production methods, which consume roughly 1 % of the world's total energy use. The use of catalysts to manipulate the required electron and proton transfer reactions with low energy input is also a chemical challenge that requires development of fundamental reaction pathways. This work presents an approach to the electrochemical reduction of N 2 into NH 3 using a coordination complex of aluminum(III), which facilitates NH 3 production at -1.16 V vs. SCE. Reactions performed under 15 N 2 liberate 15 NH 3 . Electron paramagnetic resonance spectroscopic characterization of a reduced intermediate and investigations of product inhibition, which limit the reaction to sub-stoichiometric, are also presented., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)