Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 41, Pages 16956-16961Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1109423108
Keywords
cytochrome; electron hopping; peroxidase; protein oxidation; protein radical
Categories
Funding
- National Institute of Health [National Institute of General Medical Science/National Cancer Institute Collaborative Access Team, Advanced Photon Source (APS)] [GM-41574, GM-66569, Y1-CO-1020, Y1-GM-1104]
- National Science Foundation [MCB-0843537]
- Minnesota Partnership for Biotechnology and Medical Genomics [SPAP-05-0013-P-FY06]
- US Department of Energy, Basic Energy Sciences, Office of Science [DE-AC02-06CH11357]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0843537] Funding Source: National Science Foundation
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The diheme enzyme MauG catalyzes the posttranslational modification of the precursor protein of methylamine dehydrogenase (preMADH) to complete biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Catalysis proceeds through a high valent bis-Fe(IV) redox state and requires long-range electron transfer (ET), as the distance between the modified residues of preMADH and the nearest heme iron of MauG is 19.4 angstrom. Trp199 of MauG resides at the MauG-preMADH interface, positioned midway between the residues that are modified and the nearest heme. W199F and W199K mutations did not affect the spectroscopic and redox properties of MauG, or its ability to stabilize the bis-Fe(IV) state. Crystal structures of complexes of W199F/K MauG with preMADH showed no significant perturbation of the MauG-preMADH structure or protein interface. However, neither MauG variant was able to synthesize TTQ from preMADH. In contrast, an ET reaction fromdiferrous MauG to quinone MADH, which does not require the bis-Fe(IV) intermediate, was minimally affected by the W199F/K mutations. W199F/K MauGs were able to oxidize quinol MADH to form TTQ, the putative final two-electron oxidation of the biosynthetic process, but with k(cat)/K-m values approximately 10% that of wild-type MauG. The differential effects of the W199F/K mutations on these three different reactions are explained by a critical role for Trp199 in mediating multistep hopping from preMADH to bis-Fe(IV) MauG during the long-range ET that is required for TTQ biosynthesis.
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