This proposal will describe how specific enzymes control the transfer of reactive electrons and the activation of molecular oxygen, while minimizing oxidative damage. This is central to cell development, health and survival. This research project includes studies of enzyme reaction mechanisms, protein structure-function relationships, protein-protein interactions, protein post- translational modification, and mechanisms of long range biological electron transfer. Kinetic, biochemical, spectroscopic and structural studies together with site-directed mutagenesis will be used in these studies. This proposal focuses on the mechanism of biosynthesis of the protein- derived cofactor, tryptophan tryptophylquinone (TTQ), and the structure and function of a novel di-heme enzyme MauG which catalyzes the oxygenation and cross-linking of specific tryptophan residues during TTQ biogenesis in methylamine dehydrogenase (MADH). The substrate for MauG is a 119-kDa precursor protein of MADH with mono-hydroxylated 2Trp57 and no cross- link. MauG catalyzes the 6-electron oxidation of the substrate that results in the second oxygenation of 2Trp57, cross-linking of 2Trp57 and 2Trp108, and oxidation of the quinol product of the first two reactions to form oxidized TTQ. These studies will describe a new biological mechanism for oxygen activation and factors that make specific amino acid residues in proteins susceptible to oxidative modification. The results will provide insight for development of strategies to introduce novel catalytic sites into proteins and manipulate the functions of enzyme-bound hemes, as well as provide clues as to how one might mitigate naturally occurring oxidative damage to proteins. Ongoing mechanistic studies of biological electron transfer (ET) in the MADH-amicyanin-cytochrome c-551i protein complex will be extended and new ET studies will be initiated with MauG. Defining the mechanisms of long range electron transfer reactions will enhance our understanding of the fundamental processes of respiration and intermediary metabolism at the molecular level. A fundamental understanding of the mechanisms of control of biological ET reactions will provide insight into how defective protein ET leads to production of reactive oxygen species and free radicals both of which are associated with many disease states, oxidative stress and aging.

Public Health Relevance

Project Narrative: Reactive oxygen species and free radicals, which are produced as by-products of biological electron transfer and oxygen metabolism, cause non-specific oxidative damage to cell components that causes mitochondrial myopathies, many disease states, oxidative stress and aging. However, free radicals and reactive oxygen species are also required for, and used productively in biosynthetic processes. This proposal will elucidate how specific enzymes control the transfer of reactive electrons and the activation of molecular oxygen, while minimizing oxidative damage.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM041574-25
Application #
8309196
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1988-08-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
25
Fiscal Year
2012
Total Cost
$319,759
Indirect Cost
$99,236
Name
University of Central Florida
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
150805653
City
Orlando
State
FL
Country
United States
Zip Code
32826
Dow, Brian A; Davidson, Victor L (2016) Converting the bis-FeIV state of the diheme enzyme MauG to Compound I decreases the reorganization energy for electron transfer. Biochem J 473:67-72
Sehanobish, Esha; Williamson, Heather R; Davidson, Victor L (2016) Roles of Conserved Residues of the Glycine Oxidase GoxA in Controlling Activity, Cooperativity, Subunit Composition, and Cysteine Tryptophylquinone Biosynthesis. J Biol Chem 291:23199-23207
Roessler, Christian G; Agarwal, Rakhi; Allaire, Marc et al. (2016) Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography. Structure 24:631-40
Ma, Zhongxin; Williamson, Heather R; Davidson, Victor L (2016) A Suicide Mutation Affecting Proton Transfers to High-Valent Hemes Causes Inactivation of MauG during Catalysis. Biochemistry 55:5738-5745
Sehanobish, Esha; Campillo-Brocal, Jonatan C; Williamson, Heather R et al. (2016) Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis. Biochemistry 55:2305-8
Ma, Zhongxin; Williamson, Heather R; Davidson, Victor L (2016) Mechanism of protein oxidative damage that is coupled to long-range electron transfer to high-valent haems. Biochem J 473:1769-75
Dow, Brian A; Davidson, Victor L (2015) Characterization of the free energy dependence of an interprotein electron transfer reaction by variation of pH and site-directed mutagenesis. Biochim Biophys Acta 1847:1181-6
Shin, Sooim; Feng, Manliang; Li, Chao et al. (2015) A T67A mutation in the proximal pocket of the high-spin heme of MauG stabilizes formation of a mixed-valent FeII/FeIII state and enhances charge resonance stabilization of the bis-FeIV state. Biochim Biophys Acta 1847:709-16
Dow, Brian A; Tatulian, Suren A; Davidson, Victor L (2015) Use of the amicyanin signal sequence for efficient periplasmic expression in E. coli of a human antibody light chain variable domain. Protein Expr Purif 108:9-12
Sehanobish, Esha; Chacón-Verdú, María Dolores; Sanchez-Amat, Antonio et al. (2015) Roles of active site residues in LodA, a cysteine tryptophylquinone dependent ε-lysine oxidase. Arch Biochem Biophys 579:26-32

Showing the most recent 10 out of 48 publications