Environmental Molecular Sciences Laboratory

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Using High-Resolution Mass Spectrometry, NMR and Computation to Elucidate Biomolecular Structural Features that Enable Efficient

Monday, October 1, 2018
Principal Investigator: 
Yi Lu
Lead Institution: 
University of Illinois at Urbana-Champaign
Project ID: 

This proposal addresses a major under-explored area of research in our understanding of foundational principles for predictive biology central to energy production, i.e., the role of post-translational modifications in oxygen reduction reaction (ORR) catalysis in fuel cells. Recent studies have demonstrated that heme-Cu oxidase (HCO), a terminal oxidase in respiration that reduces ~90% of the O2 in the biosphere to water, is a superior ORR catalyst than the best Pt-based catalyst, because HCO catalyzes the ORR using earth-abundant metal ions (Fe and Cu) and at much lower overpotentials. Critical to this HCO function is the presence of a post-translational His-Tyr cross-link in the active site, whose formation and role in ORR catalysis is poorly understood, because HCO is a large, membrane-bound protein containing multiple metal centers, making it extremely difficult to use methods such as mass spectrometry (MS), NMR and computation to study this important issue. While small molecular models of HCO have been synthesized, no model has observed formation of the cross link, and thus cannot offer insight into how His-Tyr is formed.
To overcome these limitations, we have developed a novel biosynthetic approach wherein we have used protein engineering techniques to reproduce the active site structure of HCO in a small, stable, well-characterized and easy-to-produce protein, myoglobin (Mb). Using this approach that is currently supported by the BER Bioenergy Center at UIUC, we have developed a protein model that structurally and functionally mimics the HCO active site, and, remarkably, displays catalytic activity comparable to native HCO. We have defined the role of the Tyr in HCO through incorporation of unnatural Tyr analogues and have answered a decades-old question relating to the chemical preference of copper over iron in HCO activity.
Moving forward, our model will prove uniquely insightful in determining the structural features responsible for formation of the rare post-translational His-Tyr crosslink found in HCOs, as well as the mechanistic role of the crosslink in efficient ORR activity. This direction is motivated by exciting results from pilot studies using high-resolution MS at EMSL that provide interesting evidence for the His-Tyr crosslink in G65Y-CuBMb and an unprecedented His-His crosslink in F33Y-CuBMb. NMR results obtained at EMSL are also encouraging. Encouraged by these results, we have put together a collaborative team that includes experts in MS, NMR and computations to carry out more systematic studies of the structural features and protein dynamics responsible for the His-Tyr and His-His crosslink formations using the unique high-resolution MS, NMR and computation capabilities at EMSL. Specifically, we plan to use this system to elucidate 1) the structural features and conditions responsible for the formation of His-Tyr and His-His crosslinks using high resolution MS; 2) the structures of the post-translational modifications and structural features, including the protein dynamics, required for such formation using NMR; and 3) mechanisms of the crosslinks and protein dynamic responsible for such formation using computation.
Results from this proposed study will provide a powerful understanding of principles for post-translational modifications and its roles in ORR catalysis, which has thus far been recalcitrant to study without resources such as those available and/or unique at EMSL. The project will advance our knowledge of the structural features and mechanisms responsible for post-translational modifications and their roles in conferring and fine-tuning ORR catalysis. The resulting principles will allow us to design smaller, cheaper, more robust catalysts for fuel cells that use earth abundant metal ions such as iron and copper (instead of platinum) and possess lower over-potentials.