Article pubs.acs.org/IC
Hiding in Plain Sight: The Bimetallic Magnesium Covalent Bond in Enzyme Active Sites Lalith Perera,† William A. Beard,† Lee G. Pedersen,*,†,‡ and Samuel H. Wilson*,† †
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, North Carolina 27709-2233, United States ‡ Department of Chemistry, University of North Carolina at Chapel Hill, P.O. Box 3290, Chapel Hill, North Carolina 27517, United States S Supporting Information *
ABSTRACT: The transfer of phosphate groups is an essential function of many intracellular biological enzymes. The transfer is in many cases facilitated by a protein scaffold involving two closely spaced magnesium “ions”. It has long been a mystery how these “ions” can retain their closely spaced positions throughout enzymatic phosphate transfer: Coulomb’s law would dictate large repulsive forces between these ions at the observed distances. Here we show, however, that the electron density can be borrowed from nearby electron-rich oxygens to populate a bonding molecular orbital that is largely localized between the magnesium “ions”. The result is that the Mg−Mg core of these phosphate transfer enzymes is surprisingly similar to a metastable [Mg2]2+ ion in the gas phase, an ion that has been identified experimentally and studied with high-level quantum-mechanical calculations. This similarity is confirmed by comparative computations of the electron densities of [Mg2]2+ in the gas phase and the Mg−Mg core in the structures derived from QM/MM studies of high-resolution X-ray crystal structures. That there is a level of covalent bonding between the two Mg “ions” at the core of these enzymes is a novel concept that enables an improved vision of how these enzymes function at the molecular level. The concept is broader than magnesiumother biologically relevant metals (e.g., Mn and Zn) can also form similar stabilizing covalent Me−Me bonds in both organometallic and inorganic crystals.
I. INTRODUCTION
Observations we wish to understand in the present work include the following: (1) Why are relatively small divalent metal cations arranged so close to each other in the active site (e.g., the Mg−Mg distance in these systems has been found to be as short as 2.42 Å4)? (2) Why is it that Mg ions in bimetallic dicationic interactions observed in crystallographic structures of enzyme active sites do not occupy pseudo-symmetry-related octahedral coordination positions about the individual metals? Instead, metal ions in numerous structures obtained in the last 25 years interact through the faces of opposing coordination octahedra, as shown in Figure 1. (3) What role does the metastable covalent bond between the metal ions (established below) play in enzyme catalysis?
Bimetallic magnesium catalytic active sites involving phosphate group transfer are widespread in nature. They are essential in DNA and RNA polymerases,1 reverse transcriptases,2 DNA and RNA endo- and exonucleases,3 terminases,4 and certain kinases (CDK2).5 It has been common to think that these active-site metals function only as charged ions in stabilizing other activesite components. We present an alternate viewa view that arises from examining the quantum-mechanical nature of the “hidden” covalent metal−metal bond in the bimetallic active site and the relationship to homologous gaseous bimetallic dications. This alternate view is found to be remarkably consistent with earlier studies6 of bimetallic magnesiumcontaining compounds, for which each Mg has an additional electron and a formal covalent bond between the dications is evident. © XXXX American Chemical Society
Received: September 9, 2016
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DOI: 10.1021/acs.inorgchem.6b02189 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Figure 2. Standard molecular orbital electron configurations of the outer electrons of relevant divalent metal dications for the first four rows of the periodic table. Energy levels and their spacing have been adjusted for readability. The “*” superscript of the orbital label designates antibonding. He2, Be2, Mg2, and Ca2 (Mn2) have a net formal bond order of zero, whereas their dications have a net formal bond order of 1. Figure 1. The DNA polymerase β active site (PDB ID 2FMS).7 Each metal ion has octahedral coordination of oxygen (red) ligands. The Mg−Mg bond is indicated by a dotted line. The catalytic Mg (Mg(c)) is shown in magenta and the nucleotide-binding Mg (Mg(n)) in orange. It should be noted that the opposing metal ions do not occupy coordination positions but instead interact along the dotted line.
II. METHODS Quantum-Mechanical Calculations on Various Systems. Gaussian 09, version D0111 was employed to generate the total energy curves shown in Figure 3. Density functional theory calculations were performed using the B3LYP exchange−correlation functional and the 6-31G* basis set.12
The central hypothesis is that the bimetallic magnesium core in the active sites of many enzymes can be understood in terms of the quantum-mechanical properties of gas-phase bimetallic dications [Me2]2+, which have a rich history in experimental and theoretical chemical physics.8,9 In the simple quantummechanical description (bond orders, relative degree of stability, bond distances, magnetic properties) of the homonuclear diatomic molecules of the light elements (H2 through Ne2), the electron configuration of the diatom is displayed as an energy diagram from which the atomic orbitals of two adjacent metal atoms interact to form bonding and antibonding molecular orbitals (MOs). When the existing count of electrons in the diatom is fed into the MO energy diagram starting at the bottom, the result is an MO description of the diatom (e.g.,10 H2: (σ1s)2, He2: (σ1s)2(σ1s * )2, ..., Ne2: (σ1s)2···(σ2p * )2). By this procedure, one finds that He2, Be2, and Ne2 are predicted to not naturally exist, O2 and B2 are predicted to have triplet ground states, and the bond orders for H2, C2, and N2 are predicted to be in the order of 1:2:3. All of these features are experimentally realized. We can also apply this approach to the next period of the periodic table (Na2, Mg2, ..., Ar2). When the same concepts are employed, one finds that Mg2 (same column of the periodic table as Be2) is predicted to not exist, whereas [Mg2]2+ is predicted to exist (gas-phase) with a simple MO bond description of ([Ne2](σ3s)2)2+ and a covalent bond order of 1 (Figure 2). The purpose of this work is to seek answers to the questions raised in the early part of the Introduction that investigate the involvement of divalent bimetallic cations in enzyme catalysis. We have primarily selected the active site of DNA polymerase β, which contains two magnesium ions, as our test system since it has been the subject of a large number of structural and theoretical evaluations. In the present study, we have used quantum mechanics (QM) and quantum mechanics/molecular mechanics (QM/MM) calculations along with QM charge and electron density evaluations to characterize the behavior of the closely spaced metal ions residing in a heavily ionic environment that may foster a pseudocovalent bond between the metal ions.
Figure 3. Energy vs metal ion separation distance (R) for various [Mea−Meb]2+ systems. The curves were adjusted to converge at large R. Calculation details are given in Methods. The multiplicities of the Mn complexes are given in parentheses. Computation of Pre- and Postcatalytic Geometry Representations for DNA Polymerase β and the Charges of the Atoms in the Active Site. With the crystallographic structures of gapped DNA, dCTP, and DNA polymerase β ternary complexes (PDB ID 2FMS)7 as the starting points, molecular dynamics (MD) simulations were carried out in a completely solvated aqueous environment. Valence-filling hydrogens were added, and the systems were neutralized with counterions while preserving the positions of all of the crystal water molecules. The initial triphosphate charge was set to −3e with only one oxygen protonated in the γ-phosphate for the equilibration with the classical force field. The Amber99SB force field was used with the PMEMD module of the Amber 12 program for the trajectory calculations.13,14 Water molecules were represented by the TIP3P model.15 Long-range interactions were treated with the particle mesh Ewald method.16,17 Added water molecules were subjected to a constant pressure simulation at low temperatures (
