Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H⁺ and Na⁺ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a “direct coupling” mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na⁺ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K⁺-dependent H⁺-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PP_i analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PP_i hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg²⁺ (essential cofactor). The replacement of K⁺, the second metal cofactor, by Na⁺ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PP_i hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell’s direct coupling with conformational coupling to catalyze cation transport across the membrane.     

The Non-Gastric H+/K+ ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa

H⁺/K⁺ ATPase Type 2 is an heteromeric membrane protein involved in cation transmembrane transport and consists of two subunits: a specific α subunit (ATP12A) and a non-specific β subunit. The aim of this study was to demonstrate the presence and establish the localization of ATP12A in spermatozoa from Bubalus bubalis, Bos taurus and Ovis aries. Immunoblotting revealed, in all three species, a major band (100 kDa) corresponding to the expected molecular mass. The ATP12A immunolocalization pattern showed, consistently in the three species, a strong signal at the acrosome. These results, described here for the first time in spermatozoa, are consistent with those observed for the β₁ subunit of Na⁺/K⁺ ATPase, suggesting that the latter may assemble with the α subunit to produce a functional ATP12A dimer in sperm cells. The above scenario appeared to be nicely supported by 3D comparative modeling and interaction energy calculations. The expression of ATP12A during different stages of bovine sperm maturation progressively increased, moving from epididymis to deferent ducts. Based on overall results, we hypothesize that ATP12A may play a role in acrosome reactions. Further studies will be required in order to address the functional role of this target protein in sperm physiology.     

The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches,Interaction with the Long Noncoding HOTAIR RNA

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the ¹H^N-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, ¹H^α-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407–Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2^T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR₁₄₀ (1–140 nt) highlight that the Thr367–Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein–RNA interaction as a “fuzzy” complex.     

The β2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule

The β₂ subunit of Na⁺, K⁺-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While β₁ isoform have been shown to trans-interact in a species-specific mode with the β₁ subunit on the epithelial neighboring cell, the β₂ subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of β₂ or described its adhesion mechanism. Until now, the interactions pronounced for β₂/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether β₂ is a cell–cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell–cell aggregation, and protein–protein interaction assays. We observed that the glycosylated extracellular domain of β₂/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human β₂ subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein–protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant β₂ subunit show that the β₂ subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human β₂ subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).     

Regulation of Cytosolic pH: The Contributions of Plant Plasma Membrane H+-ATPases and Multiple Transporters

Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H⁺ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H⁺ transport activity, namely, H⁺-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H⁺ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H⁺-ATPases mediate H⁺ extrusion, whereas most members of other six proteins mediate H⁺ influx, thus contributing to cytosolic pH homeostasis by directly modulating H⁺ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H⁺-coupled substrate influx, whereas other intra-family members facilitate H⁺-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H⁺-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H⁺ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H⁺ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.     

Solid-state nuclear magnetic resonance investigation of cation siting in LiNaLSX zeolites

The location of Li⁺ and Na⁺ cations in a series of dehydrated low-silica LiNaX zeolites (LiNaLSX, framework Si/Al ratio=1.0) were characterized by ⁷Li and ²³Na magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. Depending on the Li⁺ content, up to three lines were observed in the ⁷Li MAS NMR spectra attributed to Li⁺ cations on SI′, SII and SIII sites. The ²³Na MAS NMR spectra of the pure sodium form NaLSX and of LiNaLSX samples with low Li⁺ contents contain up to five lines belong to Na⁺ cations located on SI, SI′, SII, and two different SIII′ sites. LiNaLSX zeolites containing more than 40% of Li⁺ show only a single narrow line in the ²³Na MAS NMR spectra attributed to mobile sodium cations. The populations of the different cation sites were determined from the relative line intensities of the MAS NMR spectra. Below about 70% Li⁺ exchange, lithium cations are located only on sites SI′ and SII. Between 70% and 100% Li⁺ content these sites are fully occupied by Li⁺, and the population of site SIII by Li⁺ increases. It was found that the nitrogen-adsorption capacity correlates well with the occupation of Li⁺ at site SIII.     

Niobium oxide acidity studied by proton broad-line NMR at 4 K and MAS NMR at room temperature

The quantitative study of the Brønsted acidity of niobic acid (Nb₂O₅·xH₂O) using broad-line¹H NMR at 4 K has been performed by interacting niobic acid, pretreated at 573 K under vacuum, with water molecules. The number of oxyprotonated species (H₃O⁺ and H₂O...HO species formed, unreacted acidic OH groups or excess H₂O molecules) deduced from the simulations of the broad-line¹H NMR spectra shows a continuous increase in the number of H₃O⁺ species with adsorbed water molecules. This increase may be due to a classical dilution effect or to a synergistic interaction between Brønsted and Lewis acid sites. These results are compared with those of some HY zeolites with or without framework defects.     

The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K⁺-dependent, whereas those with K117 are K⁺-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues’ contribution to the K⁺-independent pyruvate kinases using the K⁺-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K⁺-dependent and -independent mutants. K⁺-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K⁺-dependent enzymes. E117K mutant was K⁺-independent and the enzyme acquired the active conformation by a different mechanism. In the K⁺-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.     

Dehydrogenation of cyclohexanol on Cu–ZnO/SiO2 catalysts: The role of copper species

For the dehydrogenation of cyclohexanol a series of Cu–ZnO/SiO₂ catalysts with various Cu to ZnO molar ratios was prepared using the impregnation method, with the loading of copper fixed at 9.5 at.%. The catalysts were characterized by XPS, H₂–N₂O titration, BET, H₂-TPR, NH₃-TPD and XRD techniques. The results indicate that the addition of ZnO can improve the dispersion of copper species on reduced Cu–ZnO/SiO₂ (CZS) catalysts. Cu⁰ and Cu⁺ species were found on the reduced CZS catalysts surface, and the amount of Cu⁺ increased with the content of ZnO increasing. The addition of ZnO increased the acidity of the CZS catalysts. However, only Cu⁰ species can be found on the reduced Cu/SiO₂ (CS) catalyst surface. According to the reaction results, we found that the selectivity to phenol was related to the amount of Cu⁺ species, the Cu⁺ species should be the active sites for the production of phenol, the Cu⁰ is responsible for cyclohexanol dehydrogenation to cyclohexanone.     

Simultaneous Monitoring of pH and Chloride (Cl−) in Brain Slices of Transgenic Mice

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl⁻]_i) and hydrogen ([H⁺] _i) the construct, called ClopHensor, which consists of a H⁺- and Cl⁻-sensitive variant of the enhanced green fluorescent protein (E²GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H⁺]_i and [Cl⁻]_i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H⁺]_i and [Cl⁻]_i under different experimental conditions including changing of external concentrations of ions (Ca²⁺, Cl⁻, K⁺, Na⁺) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl⁻ and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl⁻ and pH.     

Altered mRNA and Protein Expression of Monocarboxylate Transporter MCT1 in the Cerebral Cortex and Cerebellum of Prion Protein Knockout Mice

The effect of a cellular prion protein (PrP^c) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrP^c in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp^−/− mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na⁺/K⁺ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp^−/− vs. WT control group, without a substantial change in the Na⁺/K⁺ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp^−/− vs. WT mice. The present study demonstrates that a lack of PrP^c leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp^−/− vs. WT mice. Our findings provide evidence that PrP^c might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.     

Effects of Water and Ion-Exchanged Counterion on the FTIR Spectra of ZSM-5. II. (Cu+–CO)-ZSM-5: Coordination of Cu+–CO Complex by H2O and Changes in Skeletal T–O–T Vibrations

The 2157 cm^–1 (strong) and 2108 cm^–1 (very weak) (CO) IR bands due to Cu⁺–CO in ZSM-5 zeolite with ¹²C and ¹³C isotopes, respectively, are reversibly red-shifted by subsequent adsorption of H₂O at 293 K. On the contrary, the locally perturbed internal (T–O–T) asymmetric stretching framework vibration [ _as ^int (TOT)(Cu⁺–CO)=965 cm^–1] is reversibly blue-shifted. The courses of the band shifts revealed notable features. Charge transfers from water to Cu⁺ ions, changes in coordination spheres of Cu⁺(CO)(H₂O) _n aqua complexes and secondary (solvent-like) effects were considered to explain the results.     

A Comparative Study to Decipher the Structural and Dynamics Determinants Underlying the Activity and Thermal Stability of GH-11 Xylanases

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11^TS. Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme–xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11^TS. By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11^TS.     

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.     

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus

Mice lacking functional thyroid follicular cells, Pax8^−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na⁺-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8^−/− mice should show deficits in the expression of Na⁺ currents and potentially also in the expression of Na⁺/K⁺-ATPases, which are necessary to maintain low intracellular Na⁺ levels. We thus compared Na⁺ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na⁺/K⁺-ATPase in wild type versus Pax8^−/− mice. Whereas the Na⁺ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na⁺ current density remained at a very low level in hippocampal neurons from Pax8^−/− mice. Pax8^−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na⁺/K⁺-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.     

Fluoride ion reactions in the acetonitrile-10%(mole) water medium. pF scale and potential-pF diagrams relative to the silver, copper and hydrogen redox systems

Ag⁺, Cu⁺, Cu²⁺, Co²⁺, UO²⁺₂, La³⁺, Fe³⁺, Al³⁺ precipitation by fluoride ions and their reaction with H⁺ ion have been studied in the acetonitrile-10%(mole) water medium. The pF⁻ buffers are positioned on a pF⁻ scale extending over about 14 unities. The potential-pF⁻ diagrams relative to the silver, copper and hydrogen systems, established in the same medium, give evidence that in contrast with water, their redox properties depend largely on the fluoride ion concentration.     

Novel Hg and Mn supramolecular complexes with a versatile building block 5-(4-pyridyl)-1,3,4-oxadiazole-2-thiolate involving in situ ligand formation

Reaction of potassium N′-(pyridine-4-carbonyl)-hydrazinecarbodithioate ([K⁺(H₂L)⁻]) with Hg^II or Mn^II inorganic salt in the presence of 1,10-phenanthroline (phen) or 4,4′-bipyridine (bipy), yields complexes [Hg(pyt)₂(phen)] (1) and {[Mn(pyt)₂(H₂O)₂]·(bipy)·(H₂O)₂}_n (2), in which the pyt ligand (Hpyt = 5-(4-pyridyl)-1,3,4-oxadiazole-2-thiol) is obtained by in situ ligand formation from the acyclic precursor [K⁺(H₂L)⁻]. Single crystal X-ray diffraction suggests that the pyt anionic ligands in 1 and 2 behave as thiolate and thione isomers, respectively, and display S- and μ-N_pyridyl, N_oxadiazole-binding fashions. Complex 1 shows a 1-D fishbone-like supramolecular array via strong aromatic stacking interactions between the discrete mononuclear coordination motifs, whereas 2 has a 2-D layered host coordination framework with the inclusion of bipy and water guests in the cavities.     

Wisteria floribunda Agglutinin-Positive Mac-2 Binding Protein as a Screening Tool for Significant Liver Fibrosis in Health Checkup

Chronic liver disease is generally widespread, and a test for screening fibrotic subjects in a large population is needed. The ability of Wisteria floribunda agglutinin-positive mac-2 binding protein (WFA⁺-M2BP) to detect significant fibrosis was investigated in health checkup subjects in this research. Of 2021 health checkup subjects enrolled in this prospective cross-sectional study, those with WFA⁺-M2BP ≥ 1.0 were defined as high risk. Liver fibrosis was evaluated using magnetic resonance elastography (MRE) in subjects with high risk. The primary outcome was the positive predictive value (PPV) of WFA⁺-M2BP for significant fibrosis (liver stiffness ≥ 2.97 kPa by MRE). This trial was registered with the UMIN clinical trial registry, UMIN000036175. WFA⁺-M2BP ≥ 1.0 was observed in 5.3% of the 2021 subjects. The PPV for significant fibrosis with the threshold of WFA⁺-M2BP at ≥1.0, ≥1.1, ≥1.2, ≥1.3, ≥1.4, and ≥1.5 was 29.2%, 36.4%, 43.5%, 42.9%, 62.5%, and 71.4%, respectively. A WFA⁺-M2BP of 1.2 was selected as the optimal threshold for significant fibrosis among high-risk subjects, and the PPV, negative predictive value, sensitivity, and specificity for significant fibrosis were 43.5%, 84.0%, 71.4%, and 61.8%, respectively. WFA⁺-M2BP ≥ 1.2 was significantly associated with significant fibrosis, with an odds ratio (OR) of 4.04 (95% confidence interval (CI): 1.1–16, p = 0.04), but not FIB-4 ≥ 2.67 (OR: 2.40, 95%CI: 0.7–8.6, p-value = 0.2). In conclusion, WFA⁺-M2BP is associated with significant fibrosis and could narrow down potential subjects with liver fibrosis. The strategy of narrowing down fibrosis subjects using WFA⁺-M2BP may be used to screen for fibrotic subjects in a large population.     

An In Situ ESR Study of Pd/H-ZSM-5 Interaction with Different Adsorbents

In situ ESR at 120–473 K permits to monitor formation of transient paramagnetic ions/complexes (isolated Pd⁺ sites; Pd⁺/H₂O; Pd⁺/C₆H₆) upon interaction of isolated Pd²⁺ cations stabilized by the H-ZSM-5 matrix with different organic compounds and gas mixtures (NO, O₂, H₂O, H₂, propene, benzene). The in situ study provides insight into the elementary steps of redox processes on isolated Pd species in H-ZSM-5 zeolite under realistic conditions. Adsorbed water stabilizes the transient paramagnetic complex and decreases the rate of Pd²⁺ to Pd⁰ reduction by H₂. Strong bonding of NO _x ligands to Pd²⁺ species suppresses the reduction of Pd(II) ions. Sorption of benzene on preoxidized Pd²⁺/HZSM-5 is accompanied by an easy formation of organic cation-radicals and of a Pd⁺/benzene complex, the paramagnetic Pd⁺/benzene structure indicating a surprisingly high resistance to further reduction to Pd⁰. Illumination of the Pd/HZSM-5 by UV-visible light causes no measurable change in the redox properties of the catalyst.