期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：22
页码：11916-11922
DOI：10.1073/pnas.1922499117
出版社：The National Academy of Sciences of the United States of America
摘要：Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both O 2 and H 2 O 2 as cosubstrates. In this study, the H 2 O 2 reaction with reduced Hypocrea jecorina LPMO9A (Cu I - Hj LPMO9A) is demonstrated to be 1,000-fold faster than the O 2 reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze–quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce Cu II - Hj LPMO9A and indicates homolytic O–O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the Cu II sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.
