Phenoxyl-copper(II) complexes: models for the active site of galactose oxidase

 The reaction of the macrocycles 1,4,7-tris (3,5-di-tert-butyl-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L¹H₃, or 1,4,7-tris(3-tert-butyl-5-methoxy-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L²H₃, with Cu(ClO₄)₂·6H₂O in methanol (in the presence of Et₃N) affords the green complexes [Cu^II(L¹H)] (1), [Cu^II(L²H)]·CH₃OH (2) and (in the presence of HClO₄) [Cu^II(L¹H₂)](ClO₄) (3) and [Cu^II(L²H₂)] (ClO₄) (4). The Cu^II ions in these complexes are five-coordinate (square-base pyramidal), and each contains a dangling, uncoordinated pendent arm (phenol). Complexes 1 and 2 contain two equatorially coordinated phenolato ligands, whereas in 3 and 4 one of these is protonated, affording a coordinated phenol. Electrochemically, these complexes can be oxidized by one electron, generating the phenoxyl-copper(II) species [Cu^II(L¹H)]^+ ·, [Cu(L²H)]^+ ·, [Cu^II(L¹H₂)]^2+ ·, and [Cu^II(L²H₂)]^2+ ·, all of which are EPR-silent. These species are excellent models for the active form of the enzyme galactose oxidase (GO). Their spectroscopic features (UV-VIS, resonance Raman) are very similar to those reported for GO and unambiguously show that the complexes are phenoxyl-copper(II) rather than phenolato-copper(III) species. Received: 10 February 1997 / Accepted: 7 April 1997     

A first model for the oxidized active form of the active site in galactose oxidase: a free-radical copper complex

 Copper(II) complexes derived from the tripodal ligand bis(3′-t–butyl-2′-hydroxybenzyl)(2-pyridylmethyl)amine (LH₂) have been studied in order to mimic the redox active site of the free radical-containing copper metalloenzyme galactose oxidase. In non-coordinating solvents such as dichloromethane, only an EPR-silent dimeric complex was obtained (L₂Cu₂). The crystal structure of L₂Cu₂ revealed a "butterfly" design of the [Cu(μOR)₂Cu] unit, which is not flattened and leads to a short Cu–Cu distance, the t–butyl groups being localized on the same side of the [Cu(μOR)₂Cu] unit. The dimeric structure was broken down by acetonitrile or by alcohols, leading quantitatively to a brown mononuclear copper(II) complex. UV-visible and EPR data indicated the coordination of the solvent in these mononuclear complexes. Electrochemical as well as chemical (silver acetate) one-electron oxidation of acetonitrile solutions of the monomeric complex led to a yellow-green solution. Based on EPR, UV-visible and resonance Raman spectroscopy, the one-electron oxidation product was identified as a cupric phenoxyl radical system. It slowly decomposes into a product where the ligand has been substituted (dimerization) in the para position of the hydroxyl group, for one of the phenolic groups. The data for the one-electron oxidized species provides strong evidence for a free-radical copper (II) complex. Received: 19 July 1996 / Accepted: 16 October 1996     

How useful are vibrational frequencies of isotopomeric O2 fragments for assessing local symmetry? Some simple systems and the vexing case of a galactose oxidase model

The tendency for mixed-isotope O₂ fragments to exhibit different stretching frequencies in asymmetric environments is examined with various levels of electronic structure theory for simple peroxides and peroxyl radicals, as well as for a variety of monocopper–O₂ complexes. The study of the monocopper species is motivated by their relevance to the active site of galactose oxidase. Extensive theoretical work with an experimental model characterized by Jazdzewski et al. (J. Biol. Inorg. Chem. 8:381–393, 2003) suggests that the failure to observe a splitting between ¹⁶O¹⁸O and ¹⁸O¹⁶O isotopomers cannot be taken as evidence against end-on O₂ coordination. Conformational analysis on an energetic basis, however, is complicated by biradical character inherent in all of the copper–O₂ singlet structures. Electronic Supplementary Material Supplementary material is available for this article at .     

Structure and mechanism of galactose oxidase: catalytic role of tyrosine 495

 The catalytic mechanism of the copper-containing enzyme galactose oxidase involves a protein radical on Tyr272, one of the equatorial copper ligands. The first step in this mechanism has been proposed to be the abstraction of a proton from the alcohol substrate by Tyr495, the axial copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to test this proposal. X-ray crystallography reveals essentially no change from wild-type other than loss of the tyrosyl hydroxyl group. Visible spectroscopy indicates a significant change in the oxidised Y495F compared to wild-type with loss of a broad 810-nm peak, supporting the suggestion that this feature is due to inter-ligand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F variant remains capable of radical formation, a fact supported by EPR measurements. Thus the significantly reduced catalytic efficiency (1100-fold lower k _cat / K _m) observed for this variant is not due to an inability to generate the Tyr272 radical. By studying azide-induced pH changes, it is clear that the reduced catalytic efficiency is due mainly to the inability of Y495F to accept protons. This provides definitive evidence for the key role of Tyr495 in the initial proton abstraction step of the galactose oxidase catalytic mechanism. Received: 17 December 1996 / Accepted: 12 March 1997     

A comparative study of galactose oxidase and active site analogs based on QM/MM Car-Parrinello simulations

A parallel study of the radical copper enzyme galactose oxidase (GOase) and a low molecular weight analog of the active site was performed with dynamical density functional and mixed quantum-classical calculations. This combined approach enables a direct comparison of the properties of the biomimetic and the natural systems throughout the course of the catalytic reaction. In both cases, five essential forms of the catalytic cycle have been investigated: the resting state in its semi-reduced (catalytically inactive) and its oxidized (catalytically active) form, A ^semi and A ^ox, respectively; a protonated intermediate B; the transition state for the rate-determining hydrogen abstraction step C, and its product D. For A and B the electronic properties of the biomimetic compound are qualitatively very similar to the ones of the natural target. However, in agreement with the experimentally observed difference in catalytic activity, the calculated activation energy for the hydrogen abstraction step is distinctly lower for GOase (16 kcal/mol) than for the mimetic compound (21 kcal/mol). The enzymatic transition state is stabilized by a delocalization of the unpaired spin density over the sulfur-modified equatorial tyrosine Tyr272, an effect that for geometric reasons is essentially absent in the biomimetic compound. Further differences between the mimic and its natural target concern the structure of the product of the abstraction step, which is characterized by a weakly coordinated aldehyde complex for the latter and a tightly bound linear complex for the former. Received 14 October 1999 · Accepted: 19 January 2000     

Studies on galactose oxidase active site model complexes: effects of ring substituents on Cu(II)-phenoxyl radical formation

Copper(II) complexes of new N₃O- and N₂O₂-donor tripodal ligands bearing one or two o-substituted phenol moieties have been synthesized as models for the galactose oxidase active site. The complexes of 2-[N-(1-methyl-2′-imidazolylmethyl)-N-(6″-methyl-2″-pyridylmethyl)-aminomethyl)]-4-methyl-6-methylthiophenol (MeSL), [Cu(MeSL)Cl], and N-(6-methyl-2-pyridylmethyl)-N,N-bis(2′-hydroxy-3′,5′-di-tert-butylbenzyl)amine (t-buL2mepy), [Cu(t-buL2mepy)(H₂O)], have been revealed by X-ray structural analysis to have a square-pyramidal structure with one and two phenolate oxygens in the basal plane, respectively. [Cu(MeSL)Cl] was converted into a Cu(II)-o-methylthiophenoxyl radical species by electrochemical or Ce(IV) oxidation. An o-methoxyphenoxyl radical in a similar complex was considerably more stable than the 2,4-di(tert-butyl)phenoxyl radical. While t-buL2mepy reacted with Cu(ClO₄)₂ to give [Cu(t-buL2mepy)(H₂O)] without disproportionation, an N2O2-donor ligand containing an o-methoxyphenol, a 2,4-di(tert-butyl)phenol, and an N-methylimidazole moiety gave a phenoxyl radical complex exhibiting the characteristic absorption peak at 478 nm as a reddish powder by the reaction with Cu(ClO₄)₂ as a result of spontaneous disproportionation. It exhibited a quasi-reversible redox wave at E_1/2=0.34 V (vs. Ag/AgCl) in CH₃CN, which is lower than the potentials of the copper complexes of various N₃O-donor ligands, and oxidized ethanol to acetaldehyde with a low turnover number.     

Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes

To elucidate the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes, we analyzed and compared the crystal structures of these enzymes, their complexes with inhibitors, and zymogens in the active site area (a total of 82 structures). In addition to the water molecule (W1) located between the active carboxyls and playing a role of the nucleophile during catalytic reaction, another water molecule (W2) at the vicinity of the active groups was found to be completely conserved. This water molecule plays an essential role in formation of a chain of hydrogen-bonded residues between the active site flap and the active carboxyls on ligand binding. These data suggest a new approach to understanding the role of residues around the catalytic site, which can assist the development of the catalytic reaction. The influence of groups adjacent to the active carboxyls is manifested by pepsin activity at pH 1.0. Some features of pepsin-like enzymes and their mutants are discussed in the framework of the approach.     

Theoretical study on electronic structures of FeOO, FeOOH, FeO(H2O), and FeO in hemes: as intermediate models of dioxygen reduction in cytochrome c oxidase

The electronic structures of heme-dioxygen complexes have been studied as intermediate models of dioxygen reduction mechanism catalyzed by the mixed valence (MV) and fully reduced (FR) cytochrome c oxidase (CcO). Dioxygen, protons and electrons were sequentially added to the heme along the proposed reaction path for the O(2) reduction mechanism. The electronic structures of [FeOO], [FeOO](-), [FeOOH](+), [FeOOH], [Fe=O, H(2)O](+), [Fe=O](+) and [Fe=O] were thoroughly investigated by using the unrestricted hybrid exchange-correlation functional B3LYP method. The additions of two protons and an electron to [FeOO] lead to the OO bond cleavage to produce a H(2)O molecule with the electron transfer from Fe(II) in heme to the OO moiety. It is shown that the intrinsic OO bond cleavage occurs by adding two protons and two electrons into the OO bond, indicating consistency with a H(2)O formation catalyzed by both MV and FR CcO. The study of the electronic structures of heme-dioxygen complexes gave the different proposals for the mechanisms of a H(2)O formation by both MV and FR CcO. For the mixed valence CcO, starting from the [FeOO] complex, the final products are single H(2)O molecule and compound I of the oxo heme. For the fully reduced CcO, the final products are single H(2)O molecule and compound II of the oxo heme which is a reduced state of the compound I.     

Proton pumping in cytochrome c oxidase: Energetic requirements and the role of two proton channels

Cytochrome c oxidase is a superfamily of membrane bound enzymes catalyzing the exergonic reduction of molecular oxygen to water, producing an electrochemical gradient across the membrane. The gradient is formed both by the electrogenic chemistry, taking electrons and protons from opposite sides of the membrane, and by proton pumping across the entire membrane. In the most efficient subfamily, the A-family of oxidases, one proton is pumped in each reduction step, which is surprising considering the fact that two of the reduction steps most likely are only weakly exergonic. Based on a combination of quantum chemical calculations and experimental information, it is here shown that from both a thermodynamic and a kinetic point of view, it should be possible to pump one proton per electron also with such an uneven distribution of the free energy release over the reduction steps, at least up to half the maximum gradient. A previously suggested pumping mechanism is developed further to suggest a reason for the use of two proton transfer channels in the A-family. Since the rate of proton transfer to the binuclear center through the D-channel is redox dependent, it might become too slow for the steps with low exergonicity. Therefore, a second channel, the K-channel, where the rate is redox-independent is needed. A redox-dependent leakage possibility is also suggested, which might be important for efficient energy conservation at a high gradient. A mechanism for the variation in proton pumping stoichiometry over the different subfamilies of cytochrome oxidase is also suggested. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Looking at enzymes from the inside out: the proximity of catalytic residues to the molecular centroid can be used for detection of active sites and enzyme-ligand interfaces

Analysis of the distances of the exposed residues in 175 enzymes from the centroids of the molecules indicates that catalytic residues are very often found among the 5% of residues closest to the enzyme centroid. This property of catalytic residues is implemented in a new prediction algorithm (named EnSite) for locating the active sites of enzymes and in a new scheme for re-ranking enzyme-ligand docking solutions. EnSite examines only 5% of the molecular surface (represented by surface dots) that is closest to the centroid, identifying continuous surface segments and ranking them by their area size. EnSite ranks the correct prediction 1-4 in 97% of the cases in a dataset of 65 monomeric enzymes (rank 1 for 89% of the cases) and in 86% of the cases in a dataset of 176 monomeric and multimeric enzymes from all six top-level enzyme classifications (rank 1 in 74% of the cases). Importantly, identification of buried or flat active sites is straightforward because EnSite "looks" at the molecular surface from the inside out. Detailed examination of the results indicates that the proximity of the catalytic residues to the centroid is a property of the functional unit, defined as the assembly of domains or chains that form the active site (in most cases the functional unit corresponds to a single whole polypeptide chain). Using the functional unit in the prediction further improves the results. The new property of active sites is also used for re-evaluating enzyme-inhibitor unbound docking results. Sorting the docking solutions by the distance of the interface to the centroid of the enzyme improves remarkably the ranks of nearly correct solutions compared to ranks based on geometric-electrostatic-hydrophobic complementarity scores.     

A critical role for the histidine residues in the catalytic function of acyl-CoA:cholesterol acyltransferase catalysis: evidence for catalytic difference between ACAT1 and ACAT2

To investigate a role for histidine residues in the expression of normal acyl-CoA:cholesterol acyltransferase (ACAT) activity, the histidine residues located at five different positions in two isoenzymes were substituted by alanine, based on the sequence homology between ACAT1 and ACAT2. Among the 10 mutants generated by baculovirus expression technology, H386A-ACAT1, H460A-ACAT1, H360A-ACAT2, and H399A-ACAT2 lost their enzymatic activity completely. A reduction in catalytic activity is unlikely to result from structural changes in the substrate-binding pocket, because their substrate-binding affinities were normal. However, the enzymatic activity of H386A-ACAT1 was restored to <37% of the level of the wild-type activity when cholesterol was replaced by 25-hydroxycholesterol as substrate. H527A-ACAT1 and H501A-ACAT2, termed carboxyl end mutants, exhibit activities of ∼96% and ∼75% of that of the wild-type. Interestingly, H425A-ACAT1 showed 59% of the wild-type activity, in contrast to its equivalent mutant, H399A-ACAT2. These results demonstrate that the histidine residues located at the active site are very crucial both for the catalytic activity of the enzyme and for distinguishing ACAT1 from ACAT2 with respect to enzyme catalysis and substrate specificity.     

Genetics and development of ocular oxidases in the mouse: evidence for a new locus (Eox-l) closely linked with the aldehyde oxidase loci on chromosome 1

Summary. Isoelectric focusing (IEF) and histochemical techniques were used to examine the genetics, postnatal development and biochemical properties of ocular oxidases (EOXs) among inbred strains of mice. The designation as EOX was made on a provisional basis, since the 'natural' substrate(s) for this enzyme have not been identified. Five major forms were resolved from adult animals, which exhibited high activity in murine lens and low activity in the cornea. An additional ocular oxidase was observed in neonatal animals. Genetic analyses demonstrated that one of these enzymes (EOX-1) is encoded by a locus (Eox-1) which is closely linked with, but distinct from, the aldehyde oxidase (Aox) gene complex on chromosome one of the mouse. These results support the proposal that ocular oxidases are distinct from the major liver AOXs in this organism.     

Spectroscopic description of an unusual protonated ferryl species in the catalase from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> and density functional theory calculations on related models. Consequences for the ferryl protonation state in catalase,peroxidase and chloroperoxidase

The catalase from Proteus mirabilis peroxide-resistant bacteria is one of the most efficient heme-containing catalases. It forms a relatively stable compound II. We were able to prepare samples of compound II from P. mirabilis catalase enriched in ⁵⁷Fe and to study them by spectroscopic methods. Two different forms of compound II, namely, low-pH compound II (LpH II) and high-pH compound II (HpH II), have been characterized by Mössbauer, extended X-ray absorption fine structure (EXAFS) and UV-vis absorption spectroscopies. The proportions of the two forms are pH-dependent and the pH conversion between HpH II and LpH II is irreversible. Considering (1) the Mössbauer parameters evaluated for four related models by density functional theory methods, (2) the existence of two different Fe–O_ferryl bond lengths (1.80 and 1.66 Å) compatible with our EXAFS data and (3) the pH dependence of the α band to β band intensity ratio in the absorption spectra, we attribute the LpH II compound to a protonated ferryl Fe^IV–OH complex (Fe–O approximately 1.80 Å), whereas the HpH II compound corresponds to the classic ferryl Fe^IV=O complex (Fe=O approximately 1.66 Å). The large quadrupole splitting value of LpH II (measured 2.29 mm s^?1 vs. computed 2.15 mm s^?1) compared with that of HpH II (measured 1.47 mm s^?1 vs. computed 1.46 mm s^?1) reflects the protonation of the ferryl group. The relevancy and involvement of such (Fe^IV=O/Fe^IV–OH) species in the reactivity of catalase, peroxidase and chloroperoxidase are discussed.     

The chemistry of the CuB site in cytochrome c oxidase and the importance of its unique His-Tyr bond

The Cu_B metal center is at the core of the active site of the heme-copper oxidases, comprising a copper atom ligating three histidine residues one of which is covalently bonded to a tyrosine residue. Using quantum chemical methodology, we have studied the Cu_B site in several redox and ligand states proposed to be intermediates of the catalytic cycle. The importance of the His-Tyr crosslink was investigated by comparing energetics, charge, and spin distributions between systems with and without the crosslink. The His-Tyr bond was shown to decrease the proton affinity and increase the electron affinity of both Tyr-244 and the copper. A previously unnoticed internal electronic equilibrium between the copper atom and the tyrosine was observed, which seems to be coupled to the unique structure of the system. In certain states the copper and Tyr-244 compete for the unpaired electron, the localization of which is determined by the oxygenous ligand of the copper. This electronic equilibrium was found to be sensitive to the presence of a positive charge 10 Å away from the center, simulating the effect of Lys-319 in the K-pathway of proton transfer. The combined results provide an explanation for why the heme-copper oxidases need two pathways of proton uptake, and why the K-pathway is active only in the second half of the reaction cycle.     

Beyond history and “on a roll”: The list of the most well‐studied human protein structures and overall trends in the protein data bank

Of the roughly 20,000 canonical human protein sequences, as of January 20, 2021, 7,077 proteins have had their full or partial, medium‐ to high‐resolution structures determined by x‐ray crystallography or other methods. Which of these proteins dominate the protein data bank (the PDB) and why? In this paper, we list the 273 top human protein structures based on the number of their PDB entries. This set of proteins accounts for more than 40% of all available human PDB entries and represent past trends as well as current status for protein structural biology. We briefly discuss the relationship which some of the prominent protein structures have with protein research as a whole and mention their relevance to human diseases. The top‐10 soluble and membrane proteins are all well‐known (most of their first structures being deposited more than 30 years ago). Overall, there is no dramatic change in recent trends in the PDB. Remarkably, the number of structure depositions has grown nearly exponentially over the last 10 or more years (with a doubling time of 7 years for proteins, obtained from any organism). Growth in human protein structures is slightly faster (at 5.9 years). The information in this paper may be informative to senior scientists but also inspire researchers who are new to protein science, providing the year 2021 snap‐shot for the state of protein structural biology.