Biosynthetic Inorganic Chemistry and its application in environmentally benign catalysis in renewable energy generation and pharmaceuticals.
Potential Tuning
Heternuclear Metalloenzymes
Unnatural Amino Acids and Non-Native Cofactors
Copper & electron transfer
(For in-depth reviews of this research, please see our publications in Nature, Inorganic Chemistry, and Angewandte Chemie)

For many years, chemical and biological systems have been developed almost independently of each other, even though they often catalyze the same types of reactions such as nitrogen fixation and methane hydroxylation. The main reason for such a separation is that they use very different approaches, each of which has its own advantages and disadvantages. While a chemical approach results in robust catalysts that are much easier and cheaper to synthesize, a biological approach produces highly efficient enantioselective enzymes that work under much milder conditions in an environmentally benign way, such as using biocompatible and biodegradable molecules and water as a solvent. A grand challenge is to design a single system that combines the benefits of both chemical and biological systems. To meet such a challenge, chemists have succeeded in making a number of biomimetic compounds that model enzymes, which is a major branch of chemical biology. Despite this tremendous progress, major challenges still remain, and it remains difficult to mimic many features of enzymes such as secondary coordination sphere tuning, enantioselectivity, and catalysis under ambient conditions in water. Few biomimetic compounds are both structural and functional models of enzymes. Even fewer compounds can replace enzymes for industrial and pharmaceutical applications.

A factor critical to success in any synthetic project is the design of ligands. The key difference between chemical and biological approaches is the choice of ligand; while a chemical approach uses organic molecules, a biological approach employs biomolecules such as proteins that are capable of endowing all of the advantageous features that can be found in enzymes. Despite these advantages, difficulties associated with biosynthesis and study--low yield and low stability of protein-based catalysis--once precluded their use in synthetic chemistry. Thanks to recent advances in biology, many proteins can be synthesized stabily in a short period of time and in high yields. We therefore believe the time is right to take advantage of biological machinery to advance inorganic coordination chemistry by using small, stable, easy-to-make, and well-characterized proteins as ligands in synthesizing novel inorganic compounds and enriching the principles of coordination chemistry. This approach, referred to as biosynthetic inorganic chemistry, has allowed us to synthesize, A) close structural and functional models of otherwise more complex metalloproteins and, through the process, offer deeper and many times unique insights into either protein structure and function or fundamental coordination chemistry, and B) novel compounds that are unprecedented in either inorganic chemistry or biology.

We focus on designing environmentally benign catalysts with applications in renewable energy generation and small molecule activation or transformation. For example, we are interested in designing metalloproteins with tunable redox potentials and their applications in many chemical and biological processes from efficient electron transfer (ET) agents in photosynthesis and respiration to catalysis in water oxidation and N2 fixation. In addition, we have designed biosynthetic models of heteronuclear metalloenzymes such as heme-copper oxidases (HCO) that are efficient biofuel cells, nitric oxide reductases (NOR) involved in the nitrogen cycle, and manganese peroxidases involved in biomass conversion. In this project, we are trying to answer key questions of why a protein containing a heme-non-heme iron center (as in NOR) is effective at 2e- reduction of NO, allowing N-N bond formation, while a protein containing a heme-copper center (as in HCO) is proficient at 4e- reduction of O2, enabling O-O bond cleavage. Finally, we are introducing unnatural amino acids and non-native cofactors , including many inorganic and organometallic catalysts, to make them water-soluble, asymmetric catalysts for applications such as synthesizing chiral intermediates in pharmaceutical drugs.

Significant recent contributions to this area

We have demonstrated that non-covalent interactions around the primary coordination spheres, such as hydrophobicity and hydrogen bonding (particularly those involving precisely positioned water), can play key roles in not only fine-tuning but sometimes also conferring functional properties of metalloproteins. Even though progressively more evidence for the importance of these non-covalent interactions has been shown in the native proteins through high resolution crystallography and NMR studies, incorporation of these features into either designed proteins or synthetic models has been rare. We have now demonstrated several examples using the biosynthetic approach:
  1. Redox potentials and electron transfer (ET) are at the heart of numerous chemical and biological functions from photosynthesis and respiration to biocatalytic nitrogen fixation and water oxidation. A long-standing issue in the field is how redox potentials of metalloproteins such as blue copper proteins can be fine-tuned over a broad range with little change to the metal-binding site or other ET properties. In addition, predictable tuning of redox active compounds over a wide range of potentials has been a significant and currently unmet challenge. In a paper published in Nature, we have demonstrated redox potential tuning of a single cupredoxin, azurin, across a 700 mV range, which is broader than that of native cupredoxins (~500 mV), through fine-tuning of three non-covalent secondary coordination sphere interactions: hydrophobicity, hydrogen-bonding, and peptide bond oxygen interactions. We further demonstrate that these features are additive, making redox potential tuning of azurin predictable and unprecedented, while introducing a new level of understanding of long-range, non-covalent interactions in tuning protein functions.
  2. Lower reorganization energies in the ET process generally increase ET rate constants and efficiency. However, rational design of ET centers to lower the reorganization energy has so far not been demonstrated. Such a task is particularly challenging for ET proteins like the blue copper protein, azurin, which have already been shown to possess very low reorganization energies in comparison to the majority of other proteins. In a paper published in PNAS, we reported an analysis of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants, where the secondary copper coordination sphere has been fine-tuned in order to span a wide range of reduction potentials, leaving the metal binding site intact. These results show that the reorganization energy of ET is indeed smaller than that of WT azurin due to an increased flexibility of the copper site.
  3. Nitric oxide reductase (NOR) is a vital enzyme whose study will impact several fields, including enzymatic mechanisms that produce and utilize NO in a variety of signal transduction pathways and understanding the greenhouse effect of N2O, generated by microorganisms. However, the field has been slowed by the lack of a good expression system to make a large amount of active enzymes as well as the lack of a high resolution crystal structure until recently. By rationally designing a structural and functional NOR mimic in myoglobin, we have contributed to the field by demonstrating the importance of one glutamate in both iron binding and NOR activity, and another glutamate in increasing the NOR activity by hydrogen bonding network, before the crystal structure of active cNOR was available. An overlay of the structures of our model NOR in myoglobin and native cNOR showed excellent agreements. Such designed proteins provides an excellent model system for understanding NOR as well as for practical applications in biological and environmental systems.
  4. While breakthroughs in protein design have been made recently, most designed enzymes contain relatively simple active site structures and display low activities with limited turnovers. Designing artificial enzymes with complex active site structures and high turnover activities will be a major step forward in this important and emerging field, because it requires sophisticated design that takes the complex protein structure into account. We have met this challenge by reporting the rational design of a functional metalloenzyme with a heterogeneous binuclear metal center that can catalyze the reduction of oxygen to water with over 1000 turnovers. In the process, we demonstrated the critical role of a tyrosine next to a histidine ligand to the CuB center of the heme copper oxidase in conferring the activity, through a hydrogen bonding network involving water. Furthermore, the fundamental importance of efficient and complete catalytic reduction of oxygen to water spans the range from biological respiration to alternative energy production in fuel cells. While many catalysts that reduce oxygen to water have been reported, a long-standing challenge is to carry out the reaction without producing reactive oxygen species (ROS), including superoxide and peroxide. ROS not only damage biomolecules in cells and components in fuel cells, but also decrease energy efficiency as ROS are a result of incomplete catalysis. In addition, catalysts that do not use expensive, precious metals, are highly desirable to minimize catalyst cost for practical applications. In this work we have designed biocatalysts that use readily available and inexpensive heme iron and copper to cleanly reduce oxygen to water with minimal release of superoxide or peroxide. Insights gained from further studies of such a designed enzyme with efficient oxygen reduction activity may lead to alternative catalysts to precious metal catalysts in fuel cells.

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