An unusual di-heme enzyme MauG catalyzes the 6-electron oxidation of a precursor methylamine dehydrogenase with a monohydroxylated 2Trp57 (preMADH) to form the mature tryptophan tryptophyl quinone (TTQ) cofactor. The reaction proceeds via three, two-electron oxidations involving the insertion of the second oxygen atom into 2Trp57, formation of the crosslink between 2Trp57 and 2Trp108, and oxidation to the quinone. The order of these modifications is unknown. MauG can use 3 moles of either H2O2 or O2 plus reducing equivalents to oxidize preMADH. Addition of stoichiometric H2O2 to MauG results in the formation of a catalytically competent bis-Fe(IV) species with one of the hemes in a Fe(IV)=O state while the other is a Fe(IV) species ligated by His and Tyr ligands. This intermediate demonstrates an unprecedented method for stabilizing a highly oxidizing species equivalent to an Fe(V). A crystal structure of the preMADH- MauG complex shows that the site of oxygen binding and activation is over 30 z from the TTQ site. Addition of excess H2O2 to the crystals results in formation of TTQ, demonstrating that the crystals are catalytically active, that each step occurs processively without dissociation of the complex, and that oxidation occurs via long range inter-protein electron transfer. One goal of this project is to structurally characterize the bis-Fe(IV) catalytic intermediate of MauG. The other objective is to characterize the order and nature of each of the 2-electron oxidation reactions occurring at the TTQ site. These biosynthetic intermediates will be generated in crystallo and structurally characterized. Mass spectrometry will also be used to confirm the results of crystallography experiments and to characterize steps involving proton transfer or radical formation, which has been implicated in the first oxidation step. These experiments promise to provide significant insight into methods of radical and high- valent oxidant stabilization within proteins as well as mechanisms of inter-protein electron transfer. These processes underpin aerobic metabolism and have been implicated in various disease states and aging, making them significant to human health.

Public Health Relevance

Cell damage due to reactive oxygen species and free radicals has been linked to aging as well as certain cancers and a host of other disease states. The unusual properties of MauG provide an excellent opportunity to enhance our understanding of the biological control of radicals and reactive oxygen species as well as mechanisms of oxygen activation and long-range inter-protein electron transfer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM097779-03
Application #
8463218
Study Section
Special Emphasis Panel (ZRG1-F04B-D (20))
Program Officer
Flicker, Paula F
Project Start
2011-05-01
Project End
2013-06-30
Budget Start
2013-05-01
Budget End
2013-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$11,457
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Shin, Sooim; Yukl, Erik T; Sehanobish, Esha et al. (2014) Site-directed mutagenesis of Gln103 reveals the influence of this residue on the redox properties and stability of MauG. Biochemistry 53:1342-9
Cheng, Zhongjun; Cheung, Peggie; Kuo, Alex J et al. (2014) A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36. Genes Dev 28:1758-71
Wilmot, Carrie M; Yukl, Erik T (2013) MauG: a di-heme enzyme required for methylamine dehydrogenase maturation. Dalton Trans 42:3127-35
Abu Tarboush, Nafez; Yukl, Erik T; Shin, Sooim et al. (2013) Carboxyl group of Glu113 is required for stabilization of the diferrous and bis-Fe(IV) states of MauG. Biochemistry 52:6358-67
Yukl, Erik T; Liu, Fange; Krzystek, J et al. (2013) Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis. Proc Natl Acad Sci U S A 110:4569-73
Yukl, Erik T; Jensen, Lyndal M R; Davidson, Victor L et al. (2013) Structures of MauG in complex with quinol and quinone MADH. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:738-43
Feng, Manliang; Jensen, Lyndal M R; Yukl, Erik T et al. (2012) Proline 107 is a major determinant in maintaining the structure of the distal pocket and reactivity of the high-spin heme of MauG. Biochemistry 51:1598-606
Yukl, Erik T; Wilmot, Carrie M (2012) Cofactor biosynthesis through protein post-translational modification. Curr Opin Chem Biol 16:54-9