Conspectus Natural metalloenzymes are often the most proficient catalysts in terms of their activity, selectivity, and ability to operate at mild conditions. However, metalloenzymes are occasionally surprising in their selection of catalytic metals, and in their responses to metal substitution. Indeed, from the isolated standpoint of producing the best catalyst, a chemist designing from first-principles would likely choose a different metal. For example, some enzymes employ a redox active metal where a simple Lewis acid is needed. Such are several hydrolases. In other cases, substitution of a non-native metal leads to radical improvements in reactivity. For example, histone deacetylase 8 naturally operates with Zn2+ in the active site but becomes much more active with Fe2+. For β-lactamases, the replacement of the native Zn2+ with Ni2+ was suggested to lead to higher activity as predicted computationally. There are also intriguing cases, such as Fe2+- and Mn2+-dependent ribonucleotide reductases and W4+- and Mo4+-dependent DMSO reductases, where organisms manage to circumvent the scarcity of one metal (e.g., Fe2+) by creating protein structures that utilize another metal (e.g., Mn2+) for the catalysis of the same reaction. Naturally, even though both metal forms are active, one of the metals is preferred in every-day life, and the other metal variant remains dormant until an emergency strikes in the cell. These examples lead to certain questions. When are catalytic metals selected purely for electronic or structural reasons, implying that enzymatic catalysis is optimized to its maximum? When are metal selections a manifestation of competing evolutionary pressures, where choices are dictated not just by catalytic efficiency but also by other factors in the cell? In other words, how can enzymes be improved as catalysts merely through the use of common biological building blocks available to cells? Addressing these questions is highly relevant to the enzyme design community, where the goal is to prepare maximally efficient quasi-natural enzymes for the catalysis of reactions that interest humankind. Due to competing evolutionary pressures, many natural enzymes may not have evolved to be ideal catalysts and can be improved for the isolated purpose of catalysis in vitro when the competing factors are removed. The goal of this Account is not to cover all the possible stories but rather to highlight how variable enzymatic catalysis can be. We want to bring up possible factors affecting the evolution of enzyme structure, and the large- and intermediate-scale structural and electronic effects that metals can induce in the protein, and most importantly, the opportunities for optimization of these enzymes for catalysis in vitro.

Histone deacetylases (HDACs) are responsible for the removal of acetyl groups from histones, resulting in gene silencing. Overexpression of HDACs is associated with cancer, and their inhibitors are of particular interest as chemotherapeutics. However, HDACs remain a target of mechanistic debate. HDAC class 8 is the most studied HDAC, and of particular importance due to its human oncological relevance. HDAC8 has traditionally been considered to be a Zn-dependent enzyme. However, recent experimental assays have challenged this assumption and shown that HDAC8 is catalytically active with a variety of different metals, and that it may be a Fedependent enzyme in vivo. We studied two opposing mechanisms utilizing a series of divalent metal ions in physiological abundance (Zn ). Extensive sampling of the entire protein with different bound metals was done with the mixed quantum-classical QM/DMD method. Density functional theory (DFT) on an unusually large cluster model was used to describe the active site and reaction mechanism. We have found that the reaction profile of HDAC8 is similar among all metals tested, and follows one of the previously published mechanisms, but the rate-determining step is different from the one previously claimed. We further provide a scheme for estimating the metal binding affinities to the protein. We use the quantum theory of atoms in molecules (QTAIM) to understand the different binding affinities for each metal in HDAC8 as well as the ability of each metal to bind and properly orient the substrate for deacetylation. The combination of this data with the catalytic rate constants is required to reproduce the experimentally observed trend in metal-depending performance. We predict Co 2+ and Zn 2+ to be the most active metals in HDAC8, followed by Fe 2+ , and Mn 2+ and Mg 2+ to be the least active. ■ INTRODUCTIONThe acetylation of lysine residues is an important reversible post-translational modification that modulates protein function, affecting a variety of cellular processes. 1−5 Proteomic surveys 6−8 have identified acetyl-lysine residues in diverse groups of proteins, including transcription factors, 9,10 cell signaling proteins, 11 metabolic enzymes (most prominently acetyl-CoA synthase 12−14 ), structural proteins in the cytoskeleton, 15,16 and HIV viral proteins. 17,18 One of the first discovered examples of lysine acetylation was that occurring in histones, 19,20 the predominant protein components of chromatin. Acetylation of histones has been linked to gene regulation: the addition of an acetyl moiety to histone lysine residues gives rise to an open chromatin structure that facilitates DNA transcription, while the removal of acetyl from histone acetyl-lysine residues is associated with a closed chromatin structure, transcriptional repression, and gene silencing. 21 The enzymes responsible for the addition and removal of acetyl groups are known as histone acetyltransferases (HATs) and histone deacetylases (HDACs) 22−26 for historical reasons, although it is now recog...

An atomistic understanding of metal transport in the human body is critical to anticipate the side effects of metal-based therapeutics and holds promise for new drugs and drug delivery designs in itself. Human serum transferrin (hTF) is a central part of the transport processes with its ubiquitous ferrying of physiological Fe(III) and other transition metals, including to tightly controlled parts of the body. There is an atomistic mechanism for the uptake process with Fe(III), but not for the release process or for other metals. This study provides initial insight into these processes for a range of transition metals (Ti(IV), Co(III), Fe(III), Ga(III), Cr(III), Fe(II), Zn(II)) through fully atomistic, extensive QM/DMD sampling and a new technique we developed to calculate relative binding affinities between metal cations and the protein. It identifies protonation of Tyr188 as a trigger for metal release, rather than protonation of Lys206 or Lys296. The study identifies difficulty of metal release from hTF as potentially related to cytotoxicity. Simulations identify a few critical interactions that stabilize the metal-binding site in a flexible, nuanced manner. Statement of SignificanceHuman serum transferrin (hTF) is a Fe(III) transport protein that may be implicated in the cytotoxicity of non-native metals like Ti(IV), Ga(III), and Al(III). However, hTF transport and especially release are not well studied for metals beyond Fe(III). In this study we computationally investigate the uptake and release mechanisms and affinities for a range of transition metals (Ti(IV), Co(III), Fe(III), Ga(III), Cr(III), Fe(II), Zn(II)). We find that the tightest binding metals of this list are Ti(IV) and Ga(III): the potentially cytotoxic ones.

Metalloproteins present a considerable challenge for modeling, especially when the starting point is far from thermodynamic equilibrium. Examples include formidable problems such as metalloprotein folding and structure prediction upon metal addition, removal, or even just replacement; metalloenzyme design, where stabilization of a transition state of the catalyzed reaction in the specific binding pocket around the metal needs to be achieved; docking to metal-containing sites and design of metalloenzyme inhibitors. Even more conservative computations, such as elucidations of the mechanisms and energetics of the reaction catalyzed by natural metalloenzymes, are often nontrivial. The reason is the vast span of time and length scales over which these proteins operate, and thus the resultant difficulties in estimating their energies and free energies. It is required to perform extensive sampling, properly treat the electronic structure of the bound metal or metals, and seamlessly merge the required techniques to assess energies and entropies, or their changes, for the entire system. Additionally, the machinery needs to be computationally affordable. Although a great advancement has been made over the years, including some of the seminal works resulting in the 2013 Nobel Prize in chemistry, many aforementioned exciting applications remain far from reach. We review the methodology on the forefront of the field, including several promising methods developed in our lab that bring us closer to the desired modern goals. We further highlight their performance by a few examples of applications.