A Personal View

Funding Information: This research was supported by the Associate Laboratory for Green Chemistry—LAQV (UIDB/50006/2020 and UIDP/50006/2020), which was financed by national funds from Fundacão para a Ciência e a Tecnologia, MCTES (FCT/MCTES). Publisher Copyright: © 2023 by the author.A story going back almost 40 years is presented in this manuscript. This is a different and more challenging way of reporting my research and I hope it will be useful to and target a wide-ranging audience. When preparing the manuscript and collecting references on the subject of this paper—aldehyde oxidoreductase from Desulfovibrio gigas—I felt like I was travelling back in time (and space), bringing together the people that have contributed most to this area of research. I sincerely hope that I can give my collaborators the credit they deserve. This study is not presented as a chronologic narrative but as a grouping of topics, the development of which occurred over many years.publishersversionpublishe

Celebratory Symposium B – Metal Centres in Supramolecular and Biological Structures Design of Artificial Enzymes Using the Metals of the Periodic Table

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Enzymatic activity mastered by altering metal coordination spheres

J Biol Inorg Chem (2008) 13:1185–1195 DOI 10.1007/s00775-008-0414-3Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite reductase, cytochrome c peroxidase and cytochrome cd1 nitrite reductase), non-heme proteins (superoxide reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

J Biol Inorg Chem (2011) 16:443–460 DOI 10.1007/s00775-010-0741-zMammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AORcatalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygenatom abstraction. Our results define a new catalytic performance for AOR—the nitrite reduction—and propose a new class of molybdenum-containing nitrite reductases. Keywords Nitrite reduction Nitric oxide formation Molybdenum Xanthine oxidase Aldehyde oxidoreductase Abbreviations AOR Aldehyde oxidoreductase DMSOR Dimethylsulfoxide reductase EPR Electron paramagnetic resonance Fe/S Iron–sulfur centre Fe/S–NO Dinitrosyl–iron–sulfur complex (MGD)2–Fe Ferrous complex of di(N-methyl-Dglucamine dithiocarbamate)(MGD)2–Fe–NO Mononitrosyl–iron complex Mo-enzymes Pterin–molybdenum-containing enzymes NaR Nitrate reductases NO Nitric oxide radical SO Sulfite oxidase XO Xanthine oxidase Introduction Molybdenum is present in a wide variety of enzymes, in both prokaryotes and eukaryotes, where it performs catalyti

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

J Biol Inorg Chem (2011) 16:183–194 DOI 10.1007/s00775-011-0753-3This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed

The electron transfer complex between nitrous oxide reductase and its electron donors

J Biol Inorg Chem (2011) 16:1241–1254 DOI 10.1007/s00775-011-0812-9Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N2OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N2OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N2OR, or an electrostatic nature, in the case of P. denitrificans N2OR and A. cycloclastes N2OR. A set of well-conserved residues on the N2OR surface were identified as being part of the electron transfer pathway from the redox partner to N2OR(Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N2OR sequence). Moreover, we built a model for Wolinella succinogenes N2OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N2OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N2OR domain is similar to that found in the other electron transfer complexes

Structural and electron paramagnetic resonance (EPR) studies of mononuclear molybdenum enzymes from sulfate-reducing bacteria

Acc. Chem. Res., 2006, 39 (10), pp 788–796 DOI: 10.1021/ar050104kMolybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. The metal atom (Mo or W) is coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. In addition, these proteins contain redox cofactors such as iron-sulfur clusters and heme groups. All of these metal cofactors are along an electron-transfer pathway that mediates the electron exchange between substrate and an external electron acceptor (for oxidative reactions) or donor (for reductive reactions). We describe in this Account a combination of structural and electronic paramagnetic resonance studies that were used to reveal distinct aspects of these enzymes

Recent advances into vanadyl, vanadate and decavanadate interactions with actin

Although the number of papers about ‘‘vanadium’’ has doubled in the last decade, the studies about ‘‘vanadium and actin’’ are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate 51V NMR signals, at 516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at 560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1 : 1 binding stoichiometry and a Kd of 7.5 mM 1. Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC50 of 68 and 300 mM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 mM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the e-ATP exchange rate (k = 6.5 10 3 s 1 and 4.47 10 3 s 1 for decavanadate and vanadyl, respectively). Finally, 1H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can regulate many cellular processes of great physiological significance

Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation

Recently reported decameric vanadate (V10) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V10 species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and decavanadate solutions, indicating that V10 and tetrameric vanadate (V4) have the same binding sites in myosin S1. Concentrations as low as 50 lM decavanadate (5 lM V10 species) induces 30% of protein cleavage, whereas 500 lM metavanadate is needed to attain the same extent of cleavage. After irradiation, V10 species is rapidly decomposed, upon protein addition, forming vanadyl (V4+) species during the process. It was also observed by NMR line broadening experiments that, V10 competes with V4 for the myosin S1 binding sites, having a higher affinity. In addition, V4 interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V10 appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V10