-
Journal of the American Chemical Society Nov 2022Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural...
Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)(X) cofactor (dmgH = dimethylglyoxime and X = N, HC, and Pr). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)(CH) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.
Topics: Metalloproteins; Crystallography, X-Ray; Escherichia coli; Circular Dichroism; Kinetics
PubMed: 36378237
DOI: 10.1021/jacs.2c08885 -
International Journal of Molecular... Jul 2019Dimethyl sulfoxide reductases (DMSO) are molybdoenzymes widespread in all domains of life. They catalyse not only redox reactions, but also hydroxylation/hydration and... (Review)
Review
Dimethyl sulfoxide reductases (DMSO) are molybdoenzymes widespread in all domains of life. They catalyse not only redox reactions, but also hydroxylation/hydration and oxygen transfer processes. Although literature on DMSO is abundant, the biological significance of these enzymes in anaerobic respiration and the molecular mechanisms beyond the expression of genes coding for them are still scarce. In this review, a deep revision of the literature reported on DMSO as well as the use of bioinformatics tools and free software has been developed in order to highlight the relevance of DMSO reductases on anaerobic processes connected to different biogeochemical cycles. Special emphasis has been addressed to DMSO from extremophilic organisms and their role in nitrogen cycle. Besides, an updated overview of phylogeny of DMSOs as well as potential applications of some DMSO reductases on bioremediation approaches are also described.
Topics: Coenzymes; Extremophiles; Iron-Sulfur Proteins; Isoenzymes; Metabolic Networks and Pathways; Metalloproteins; Molybdenum; Molybdenum Cofactors; Multigene Family; Nitrogen Cycle; Oxidation-Reduction; Oxidoreductases; Phylogeny; Pteridines; Structure-Activity Relationship; Tungsten
PubMed: 31288391
DOI: 10.3390/ijms20133349 -
Molecules (Basel, Switzerland) Aug 2022The transition element molybdenum (Mo) is an essential micronutrient for plants, animals, and microorganisms, where it forms part of the active center of Mo enzymes. To... (Review)
Review
The transition element molybdenum (Mo) is an essential micronutrient for plants, animals, and microorganisms, where it forms part of the active center of Mo enzymes. To gain biological activity in the cell, Mo has to be complexed by a pterin scaffold to form the molybdenum cofactor (Moco). Mo enzymes and Moco are found in all kingdoms of life, where they perform vital transformations in the metabolism of nitrogen, sulfur, and carbon compounds. In this review, I recall the history of Moco in a personal view, starting with the genetics of Moco in the 1960s and 1970s, followed by Moco biochemistry and the description of its chemical structure in the 1980s. When I review the elucidation of Moco biosynthesis in the 1990s and the early 2000s, I do it mainly for eukaryotes, as I worked with plants, human cells, and filamentous fungi. Finally, I briefly touch upon human Moco deficiency and whether there is life without Moco.
Topics: Animals; Coenzymes; Eukaryota; Humans; Metalloproteins; Molybdenum; Molybdenum Cofactors; Plants; Pterins
PubMed: 35956883
DOI: 10.3390/molecules27154934 -
Progress in Nuclear Magnetic Resonance... Nov 2017Magnetic resonance imaging (MRI) is a powerful technique for observing the function of specific cells and molecules inside living organisms. However, compared to optical... (Review)
Review
Magnetic resonance imaging (MRI) is a powerful technique for observing the function of specific cells and molecules inside living organisms. However, compared to optical microscopy, in which fluorescent protein reporters are available to visualize hundreds of cellular functions ranging from gene expression and chemical signaling to biomechanics, to date relatively few such reporters are available for MRI. Efforts to develop MRI-detectable biomolecules have mainly focused on proteins transporting paramagnetic metals for T and T relaxation enhancement or containing large numbers of exchangeable protons for chemical exchange saturation transfer. While these pioneering developments established several key uses of biomolecular MRI, such as imaging of gene expression and functional biosensing, they also revealed that low molecular sensitivity poses a major challenge for broader adoption in biology and medicine. Recently, new classes of biomolecular reporters have been developed based on alternative contrast mechanisms, including enhancement of spin diffusivity, interactions with hyperpolarized nuclei, and modulation of blood flow. These novel reporters promise to improve sensitivity and enable new forms of multiplexed and functional imaging.
Topics: Animals; Biophysical Phenomena; Contrast Media; Diffusion; Humans; Magnetic Resonance Imaging; Membrane Transport Proteins; Metalloproteins
PubMed: 29157492
DOI: 10.1016/j.pnmrs.2017.05.002 -
International Journal of Molecular... Dec 2022Metal chelation can provide structural stability and form reactive centers in metalloproteins. Approximately one third of known protein structures are metalloproteins,... (Review)
Review
Metal chelation can provide structural stability and form reactive centers in metalloproteins. Approximately one third of known protein structures are metalloproteins, and metal binding, or the lack thereof, is often implicated in disease, making it necessary to be able to study these systems in detail. Peptide-metal complexes are both present in nature and can provide a means to focus on the binding region of a protein and control experimental variables to a high degree. Structural studies of peptide complexes with metal ions by nuclear magnetic resonance (NMR) were surveyed for all the essential metal complexes and many non-essential metal complexes. The various methods used to study each metal ion are presented together with examples of recent research. Many of these metal systems have been individually reviewed and this current overview of NMR studies of metallopeptide complexes aims to provide a basis for inspiration from structural studies and methodology applied in the field.
Topics: Coordination Complexes; Magnetic Resonance Spectroscopy; Metals; Peptides; Metalloproteins; Ions
PubMed: 36555599
DOI: 10.3390/ijms232415957 -
Chemical Reviews Jul 2022One of the hallmark advances in our understanding of metalloprotein function is showcased in our ability to design new, non-native, catalytically active protein... (Review)
Review
One of the hallmark advances in our understanding of metalloprotein function is showcased in our ability to design new, non-native, catalytically active protein scaffolds. This review highlights progress and milestone achievements in the field of metalloprotein design focused on reports from the past decade with special emphasis on designs couched within common subfields of bioinorganic study: heme binding proteins, monometal- and dimetal-containing catalytic sites, and metal-containing electron transfer sites. Within each subfield, we highlight several of what we have identified as significant and important contributions to either our understanding of that subfield or metalloprotein design as a discipline. These reports are placed in context both historically and scientifically. General suggestions for future directions that we feel will be important to advance our understanding or accelerate discovery are discussed.
Topics: Binding Sites; Catalysis; Catalytic Domain; Electrons; Metalloproteins; Models, Molecular
PubMed: 35763791
DOI: 10.1021/acs.chemrev.1c01025 -
Current Opinion in Structural Biology Aug 2022Metalloproteins comprise at least a third of all proteins that utilize redox properties of transition metals on their own or as parts of cofactors. The development of... (Review)
Review
Metalloproteins comprise at least a third of all proteins that utilize redox properties of transition metals on their own or as parts of cofactors. The development of third generation storage ring sources and X-ray free-electron lasers with femtosecond pulses in the first decade of the 21st century has transformed metalloprotein crystallography. In the past decade, cryogenic-electron microscopy single-particle analysis, which does not require crystallization of biological samples has been extensively utilized, particularly for membrane-bound metalloprotein systems. Here, we explore recent frontiers in metalloprotein crystallography and cryogenic electron microscopy, organized for convenience under three metalloprotein-centered biological cycles, focusing on contributions from each technique, their synergy and the ability to preserve metals' redox states when subjected to a particular probe.
Topics: Cryoelectron Microscopy; Crystallization; Crystallography; Crystallography, X-Ray; Metalloproteins; X-Rays
PubMed: 35841747
DOI: 10.1016/j.sbi.2022.102420 -
Accounts of Chemical Research Apr 2016Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome...
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV-vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.
Topics: Crystallography, X-Ray; Metalloproteins; Protein Conformation; X-Ray Absorption Spectroscopy
PubMed: 26975689
DOI: 10.1021/acs.accounts.5b00538 -
Molecular Microbiology Jan 2019Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for... (Review)
Review
Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N reduction to NH is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron-molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo-free nitrogenases that are less efficient at reducing N than Mo-nitrogenase. To permit biogenesis of Mo-nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high-affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo-nitrogenase genes, while it represses production of Mo-free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo-mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo-responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs.
Topics: Ammonia; Bacteria; Biological Transport; Metalloproteins; Molybdenum; Molybdoferredoxin; Nitrogen; Nitrogen Fixation; Nitrogenase; Oxidation-Reduction; Trace Elements
PubMed: 30325563
DOI: 10.1111/mmi.14152 -
Trends in Biotechnology Jan 2018Residing at the interface of chemistry and biotechnology, artificial metalloenzymes (ArMs) offer an attractive technology to combine the versatile reaction repertoire of... (Review)
Review
Residing at the interface of chemistry and biotechnology, artificial metalloenzymes (ArMs) offer an attractive technology to combine the versatile reaction repertoire of transition metal catalysts with the exquisite catalytic features of enzymes. While earlier efforts in this field predominantly comprised studies in well-defined test-tube environments, a trend towards exploiting ArMs in more complex environments has recently emerged. Integration of these artificial biocatalysts in enzymatic cascades and using them in whole-cell biotransformations and in vivo opens up entirely novel prospects for both preparative chemistry and synthetic biology. We highlight selected recent developments with a particular focus on challenges and opportunities in the in vivo application of ArMs.
Topics: Biocatalysis; Biotechnology; Metalloproteins; Protein Engineering
PubMed: 29061328
DOI: 10.1016/j.tibtech.2017.10.003