CAREER:Structural, Spectroscopic, and Mechanistic Investigations of Native and Engineered Hemoglobin-Peroxidases

The Chemistry of Life Sciences Program funds Professor Reza Ghiladi of North Carolina State University for a CAREER award to carry out research entitled 'Structural, Spectroscopic, and Mechanistic Investigations of Native and Engineered Hemoglobin-Peroxidases.' This research involves probing the mechanism and expanding the substrate profile of dehaloperoxidase (DHP), the archetype of a potentially new and emerging class of bifunctional proteins known as the hemoglobin-peroxidases. DHP is the first globin identified to possess a biologically relevant peroxidase activity, and thus provides a unique system to explore our understanding of metalloprotein mechanism, activation, and design. Despite a number of studies on DHP that have helped to elucidate several of its mechanistic pathways, it is still not understood how this bi-functional protein can act as both a hemoglobin and a peroxidase. Biochemical assays, structural studies, and spectroscopic investigations (stopped-flow UV-visible, EPR, NMR, resonance Raman, and Mössbauer) will be used to address questions pertaining to substrate binding and activation from both the peroxidase-active ferric and globin-active oxyferrous states. As tyrosyl radicals are implicated in mediating electron transfer in the DHP-catalyzed and H2O2-dependent dehalogenation of trihalophenols, low- and high-field EPR studies will be directed towards identifying the location of protein radical formation. Mutants of DHP will also be investigated for potential application in bioremediation against nitrophenol pollutants.

Metalloproteins play a central role in all living organisms, yet the governing principles of the metal-protein interaction continue to be of interest to scientists who are looking toward building paradigms for understanding metalloprotein structure-function relationships. When taken together, the research goals outlined within this proposal take advantage of the merits of a traditional bioinorganic approach that not only enable posing and addressing the types of questions about biological systems and life processes that are of interest to chemists, but can also potentially be of importance to areas outside of chemistry. These include addressing the fundamental issues of biocatalysis, enzyme mechanism, and de novo protein engineering, thus allowing for the advancement of areas as diverse as bioremediation, chemoenzymatic synthesis, and biomass conversion for renewable biofuel production. Additionally, the broader impacts resulting from the teacher-scholar activities supported by the integration of the proposed research plan with educational endeavors include the following: i) continue to provide rural K-2 and middle school science classrooms with demonstrations, lectures, and lessons plans for teachers on the chemistry of life processes through the 'Creating STARS' outreach initiative; ii) continue the mentoring of talented, disadvantaged minority high school students for summer long research internships, using the structure-function relationship in metalloenzymes to introduce basic chemical biology concepts; iii) lead undergraduate and graduate chemistry students in interdisciplinary scientific inquiry that utilizes emerging techniques in molecular biology and spectroscopy that are becoming available to the next generation of bioinorganic chemists; iv) promote the placement of undergraduate students from low-income or outside-urbanization locations into multidisciplinary research labs as vehicles for broadening the participation of underrepresented minorities. This project is receiving co-funding from the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences.