STRUCTURAL STUDIES OF REDOX STATES OF MAUG

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The objective of this collaborative project is to structurally characterize different redox states of MauG. MauG is a highly unusual di-heme enzyme that completes the synthesis of the novel amino acid derived catalytic cofactor, tryptophan tryptophylquinone (TTQ) found in the enzyme methylamine dehydrogenase (MADH). TTQ is formed by post-translational modification of two Trp residues in the MADH beta polypeptide chain during which two atoms of oxygen are incorporated into the indole ring of betaTrp57 and a covalent bond is formed between the indole rings of betaTrp57 and betaTrp108. The natural substrate for MauG (preMADH) is a 119 kDa protein precursor of MADH with mono-hydroxylated betaTrp57 and no cross-link. MauG catalyzes a six-electron oxidation to complete TTQ biosynthesis. It can do this in a H2O2-dependent or O2/reducing equivalents-dependent reaction. The enzyme has been shown to form a unprecedented di-heme bis-Fe(IV) species that is catalytically competent. This intermediate is electronically equivalent to Fe(V), and is composed of an Fe(IV)=O with the second oxidizing equivalent residing on the Fe of the second heme. We have solved the crystal structure of MauG in complex with preMADH. This has been achieved to a resolution of 2.1 ¿, and these crystals can support catalytic turnover to form TTQ without loss of diffraction. The 5-coordinate P-heme that we expect to be the site of Fe(IV)=O formation is 32 ¿ from TTQ. The 6-coordinate E-heme lies equidistant between the P-heme and TTQ, and has a highly unusual His/Tyr axial ligand configuration that we hypothesize is required for stabilizing Fe(IV). Using XAS both in solution and in the crystal, we wish to characterize the molecular environments that can support and stabilize the unusual redox species of MauG, and enable us to make fundamental discoveries about oxygen activation by an unprecedented di-heme bis-Fe(IV) intermediate. This work would be the first XAS study of this unusual enzyme.