Mechanistic and Structural Studies of Novel Heme-Containing Enzymes

University South Carolina Research Foundation, Columbia SC

Investigators

Abstract

Intellectual Merit. Heme-iron containing peroxidases are enzymes that catalyze organic substrate oxidation while reducing hydrogen peroxide to water. Two novel heme peroxidases isolated from marine sea worms, one able to incorporate halide ions such as chloride and bromide into aromatic molecules and the other able to remove them, will be studied. The haloperoxidase, Notomastus lobatus chloroperoxidase (NCPO), halogenates phenols and is unusual in requiring an organic flavin component. Amphitrite ornata dehaloperoxidase (DHP) dehalogenates halophenols. Surprisingly, the structure of DHP is that of a globin - the same protein fold found in hemo- and myoglobin (Mb). It is the first known enzymatic globin. The unique structural properties of these two peroxidases bring into question whether they utilize a new peroxidase mechanism. A four-prong hypothesis-driven experimental plan will be followed. First, rapid kinetics experiments will probe the mechanism of DHP and NCPO catalysis and directly determine whether the traditional or an alternative mechanism is employed. Second, the ability of DHP and NCPO to carry out standard peroxidase reactions will be extensively tested in order to advance our understanding of DHP and NCPO function. Third, designed DHP and Mb mutants will be prepared to probe the mechanistic role of specific amino acids near the DHP heme and explore factors in the heme/globin environment that enhance DHP function relative to Mb. Fourth, NCPO will be cloned, expressed and structurally characterized. Small NCPO crystals have been obtained. NCPO is a complex multi-subunit hemoflavoenzyme; the roles of its subunits are unknown. Such enzymes with peroxidase activity have not yet been reported. Studies of its structure and mechanism will provide new information about halogenation in higher organisms. Investigations of complex multi-subunit-containing systems, such as those proposed herein, are at the cutting edge of mechanistic enzymology. Overall, the two novel peroxidases challenge the current view of how peroxidases function. The research will probe how nature has redesigned small globin-fold proteins into peroxidases and thereby will extend the boundaries of our understanding of structure/function relations in such enzymes. Broader Impacts. The research combines the diverse disciplines of chemistry, molecular genetics and evolution, structural biology, and spectroscopy, and promotes interaction among chemistry and biology faculty and students via regular meetings of the three research groups. Students, from high school to post-graduate, will be trained. Undergraduates will continue to make significant research contributions. The three PIs have strong records training underrepresented minorities recently including two African-American students. One of the PIs directs SCienceLab, an outreach program for middle/high school science teachers and students. A specific 'Enzyme-Catalyzed Toxic Cleanup' SCiLab designed around biological dehaloperoxidases will provide a daylong hands-on, inquiry-based lab experience and convey the excitement of interfacing biological, chemical and environmental research. The goal is to show students how biological catalysts might be used in bioremediation technology. Moreover, the research results will continue to be used teaching graduate enzymology, crystallography and metallobiochemistry courses. The fundamental chemical/biochemical progress achieved may lead to the development of applications in the broader areas of biotechnology and bioremediation and will impact related ecological and environmental research.