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：2018
卷号：115
期号：28
页码：7308-7313
DOI：10.1073/pnas.1807027115
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus ( Rma ) cytochrome c . High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in Rma cytochrome c has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.
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