Nitrogen is a critical component of DNA and proteins and is essential for all forms of life. However, most organisms cannot make use of the nitrogen gas (N2) that makes up 78% of our atmosphere. Nitrogen gas must be "fixed" to reduced forms such as ammonia (NH3) before it can be metabolized by plants and animals. This process is accomplished in nature by bacteria that live symbiotically with plants. Synthetic nitrogen fixation, performed at high temperatures and high pressures via the Haber-Bosch process, is central to the production of fertilizers, allowing the planet to sustain far larger populations than was possible prior, at the cost of 1.5% of the planet's total energy use. Nitrogenases are molybdenum-containing proteins employed by nitrogen-fixing bacteria to accomplish nitrogen fixation in water at ambient temperature and pressure, in contrast to the harsh and energy-intensive conditions of the Haber-Bosch process. Nitrogenases are also capable of reacting with carbon dioxide (i.e. conducting carbon sequestration) and carbon monoxide, reducing them to molecules that can be used as liquid fuels and feedstock chemicals. However, nitrogenases are large proteins (approximately 2000 amino acids), which prevents their application as isolated proteins. In this work, the investigators will develop synthetic proteins, only 2%-4% of the size of nitrogenase, that can bind molybdenum and react with molecules in a manner analogous to that of nitrogenases. The basis of this work is the development of novel amino acids that can both bind to molybdenum and donate electrons to the metal to engage in reaction chemistry at the molybdenum metal center, combined with computational protein design to allow the incorporation of these unnatural amino acids within a well-defined and stable protein structure that will support strong metal binding and reaction chemistry. These designed molybdenum proteins will be analyzed and characterized for their structure and ability to conduct reaction chemistry, both to understand how native molybdenum proteins can conduct important reactions and as a first step toward their potential use in processes critical to problems in energy use. This work will address critical processes central to the nitrogen cycle, the carbon cycle, energy, and sustainability, providing new insights into processes of broad fundamental importance. In the long term, this work could lead toward novel sustainable solutions to reduce energy use and synthesize feedstock chemicals from non-petroleum sources. This work will train undergraduate and graduate students in highly multidisciplinary methods, including computational protein design, peptide synthesis and characterization, organic synthesis on functional-group rich substrates, training them for integrated multidisciplinary science of the 21st century.
Nitrogen fixation, the reduction of atmospheric nitrogen to ammonia, is one of the most significant processes on the planet. Nitrogen fixation is accomplished by bacteria containing the enzyme nitrogenase, a large (~2000 amino acids) enzyme containing molybdenum or vanadium and a unique iron-sulfur cluster. The accomplishment of this ancient process under mild conditions has to date never been achieved in proteins outside the native nitrogenase proteins. As inspiration, catalytic nitrogen fixation has been demonstrated using synthetic molybdenum complexes with small molecule ligands. Toward the goal of developing redox-active proteins capable of reaction chemistry similar to nitrogenase, the team will develop a synthetic molybdenum metalloprotein containing novel redox-active amino acids capable of greater electron donor ability than the native 20 amino acids. Nitrogenases and synthetic analogues accomplish dinitrogen reduction (a six-electron, six-proton process) in part due to the multiple oxidation states readily available to molybdenum (Mo(III) to Mo(VI)), plus the presence of strong electron donor ligands, including the iron-sulfur-carbide cluster. Nitrogenases and organomolybdenum complexes can also reduce electronically related compounds such as diimides (including diazine HN=NH), hydrazines (H2N-NH2), and cyanide. This work aims to develop new approaches to allow the reduction of simple compounds using synthetic proteins and provide fundamental insights into the design of catalytically active proteins. The investigators will design synthetic proteins with redox-active side chains that are not found in naturally occurring proteins and readily bind to molybdenum, vanadium and tungsten, allowing multi-electron reductions of dinitrogen and related molecules. The metal-binding properties of these designed proteins will be characterized in different metal redox states using electrochemical, biophysical, and structural methods. The designed redox-active metalloproteins will be examined for reactivity toward reduction of a series of pi-bonded compounds.
This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.