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Title:Understanding structure-function relations in heme-copper oxidase using myoglobin-based enzyme models
Author(s):Bhagi, Ambika
Director of Research:Lu, Yi
Doctoral Committee Chair(s):Lu, Yi
Doctoral Committee Member(s):Gennis, Robert B.; Hammes-Schiffer, Sharon; Gewirth, Andrew A.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Abstract:Heme-copper oxygen reductases (HCOs) are respiratory enzymes that utilize a heme-copper center to perform the four-electron reduction of oxygen to water. The HCOs share significant structural and sequence homology to another class of metalloenzyme, nitric oxide reductase (NOR), that in turn, utilize a heme-nonheme diiron center to catalyze the two electron reduction of nitric oxide to nitrous oxide during biological denitrification processes. The homology between these two classes of enzymes is not just structural but also functional: the NORs can reduce oxygen completely to water and vice-versa. In this work, we have probed these similarities, yet certain crucial differences within the structure and function of HCOs and NORs, by using their simpler-myoglobin (Mb)-based enzymatic models.To begin with, we have attempted to understand the role of nonheme metal towards oxygen activation and reduction in HCOs. While HCOs and NORs have evolved from a common ancestral protein, copper is specifically chosen (over iron) as the nonheme metal in the catalytic center of HCOs. An elegant method to elucidate this choice would be to swap the copper in HCOs with iron or other nonheme metals and probe the resulting changes in oxygen reduction reactivity. However, doing so has been impossible in HCOs due to the inherent complexity of its membranous structure. As an alternate, we have used a myoglobin mutant (FeBMb) that not only possesses a heme-nonheme metal center similar to HCOs, but can also bind copper, iron and their redox-inactive analogue zinc at its nonheme metal center. Oxygen reduction enzymatic assays reveal that Fe-FeBMb and Cu-FeBMb exhibit 11-fold and 30-fold enhancements in oxidase activity, respectively, as compared to Zn-FeBMb suggesting the unequivocal role of nonheme metal as an electron donor to oxygen. Moreover, higher reduction potential of copper, as well as the enhanced weakening of O-O bond from the higher electron density in the d-orbital of copper are central to its higher oxidase activity as against iron. Overall, this work resolves a long-standing question in bioenergetics, and renders a chemical-biological basis for designing future oxygen reduction catalysts. Moving on, we have focused on the differences in the reduction potential (E°’) of catalytic heme iron in HCOs and NORs. While most HCOs exhibit significantly high heme E°’ values (+180 to +365 mV), NORs typically exhibit heme E°’ in the lower range (-170 to 70 mV). To understand this difference and its impact on enzymatic activity (if any), we have systematically tuned heme E°’ values of Mb-based HCO and NOR mimics using two strategies: (1) modulation of H-bonding interactions of heme ligands and (2) use of different heme types. The resulting HCO/NOR mimics display ~ 200 mV variation in heme E°’ values. Detailed kinetic, electrochemical and theoretical studies on the HCO mimics reveal that higher heme E°’ favors fast oxygen binding and electron transfer to its catalytic center, both of which are required for fast and efficient oxygen reduction reaction. The low heme E°’ of NORs, on the other hand, helps them maintain required electron density on the heme-bound NO for efficient N-N coupling to form N2O. Overall, these results show how Nature has efficiently fine-tuned E° in metalloproteins to control their substrate affinities, electron transfer rates and overall enzymatic activities. In particular, heme proteins exhibit a wide variety of heme E° (spanning over 1 V), and understanding the reasons behind the same and associated implications on their enzymatic activity will help us understand these proteins in greater detail.
Issue Date:2016-04-20
Rights Information:Copyright 2016 Ambika Bhagi
Date Available in IDEALS:2016-07-07
Date Deposited:2016-05

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