Administrative Supplement for Equipment

Research Strategy Summary of Parent Award: Enzymes with complex metallocofactors in their active sites catalyze myriad transformations relevant to human health and disease. Understanding their reaction mechanisms requires molecular-level characterization of their resting states and intermediate states, and metal-specific

spectroscopic techniques are especially useful in this endeavor. However, the high nuclearity of many metallocofactors can limit the usefulness of such techniques; the signals arising from multiple metal sites can be challenging to resolve, especially in mixtures of reaction intermediates. Moreover, it is often

impossible to map the rich spectroscopic information onto the geometric structure, and this severely limits our understanding of the chemical bonding—and therefore the reactivity—of complex metallocofactors. We propose to address these challenges by developing methods for modifying the isotopic and elemental

compositions of complex metallocofactors, in particular the nitrogenase catalytic cofactors. Nitrogenases are responsible for supplying a significant portion of the fixed nitrogen on the planet, and they therefore play an important role in maintaining a healthy and growing human population. Their catalytic cofactors

are among the most complex in Nature, and as a result their reaction mechanisms have been especially difficult to characterize. To overcome these challenges and gain new insights into the mechanism of biological nitrogen fixation, we will develop chemical methods for precisely altering the isotopic and

elemental composition of nitrogenase cofactors. Our approach is to discover mild protocols for removing specific Fe sites in nitrogenase cofactors and subsequently replacing them with 57Fe. The site-selectivity of the label will allow for the electronic structure (as elucidated spectroscopically) to be connected to the

geometric structure (as defined crystallographically), and will thereby provide unprecedented insights into the chemical bonding and reactivity of nitrogenase cofactors. Studies of these cofactors in both their resting states and intermediate states comprise the heart of the proposal. We will also extend the site-

selective 57Fe labeling protocol to incorporating different metals into specific sites of nitrogenase cofactors. This will yield artificial metalloenzymes that will serve as mechanistic probes with potentially unique properties and/or reactivity. Completion of this project will provide unprecedented mechanistic

insights into biological nitrogen fixation and will articulate concepts and protocols for rendering complex metallocofactors as mechanistically tractable as mononuclear active sites. Justification for proposed equipment: The proposed modern ultracentrifuge would replace our current ultracentrifuge (a decades-old Beckman Coulter L70, heretofore referred to as the “Old Ultracentrifuge”),

which was listed in the Equipment section of our grant proposal submitted in 2021. We first describe the scientific relevance of an ultracentrifuge with respect to this project, and then the reasons for replacing the Old Ultracentrifuge with a modern ultracentrifuge. Scientific necessity for a functioning ultracentrifuge. The project is absolutely dependent on large-scale

overproduction of nitrogenase proteins from Azotobacter vinelandii (Av), and this can only be accomplished by conducting large-scale cell growths and protein purifications. The extraction of nitrogenase proteins from Av cells (50-150 g) requires cell lysis and clarification of the cell lysate in an ultracentrifuge; without this step, particles in the lysate would clog the chromatography resins, rendering

protein purification nearly impossible. Thus, an ultracentrifuge is critical for cell lysis. Indeed, when the Old Ultracentrifuge is broken, the project grinds to a halt because every experiment requires access to nitrogenase proteins (e.g., the Mo nitrogenase) and/or nitrogenase-protein-derived cofactors (e.g., FeMo-

co, the catalytic cofactor of the Mo nitrogenase, which is extracted from the Mo nitrogenase). The specific aims that would be affected without an ultracentrifuge are: 1. Aim 1. This Aim cannot be undertaken because it relies on the large-scale isolation of nitrogenase cofactors. 2. Aim 2. This Aim cannot be undertaken because it relies on the large-scale isolation of nitrogenase

cofactors as well as the apo-proteins in which they are reinserted. 3. Aim 3. This Aim cannot be undertaken for the same reasons as articulated for Aim 2. Simply put: an ultracentrifuge is one of a few critical pieces of equipment, without which the project cannot be pursued. Rational for purchasing a modern ultracentrifuge. My lab inherited the Old Ultracentrifuge from Prof.

Stephen Lippard, who purchased this equipment many years ago. Because of its age and because we use it frequently, it breaks several times per year. This has not significantly slowed down our research progress because it is typically repaired within a few days by a talented technician from Beckman Coulter.

However, Beckman Coulter recently informed us that they have stopped making replacement parts and we will therefore no longer be able to source them. As such, Beckman Coulter is not guaranteeing the repairability of the Old Centrifuge, which means that it is essentially obsolete; soon (probably within a

year), the Old Centrifuge will break and will remain permanently broken. This will be catastrophic for our research project for the reasons described above. Replacing the Old Centrifuge with a modern centrifuge would solve this problem since a modern centrifuge would be serviceable now and for the remainder of the project. Additionally, it would likely

break far less frequently, and thus would result in less down-time in our research efforts. Note that we are proposing to purchase a modern ultracentrifuge (Beckman Optima XE) that has essentially the same features as the Old Ultracentrifuge. The Optima XE is not only their best-selling model, it’s also their most economical ultracentrifuge, and as such, our proposal to purchase this piece of

equipment is financially conservative. Future costs. There will be no additional costs associated with the new ultracentrifuge as any repairs will be covered under a service contract.