A Distal Loop Controls Product Release and Chemo- and Regioselectivity in Cytochrome P450 Decarboxylases.
José A AmayaCooper D RutlandNicholas LeschinskyThomas M MakrisPublished in: Biochemistry (2017)
Cytochrome P450 OleT utilizes hydrogen peroxide (H2O2) to catalyze the decarboxylation or hydroxylation of fatty acid (FA) substrates. Both reactions are initiated through the abstraction of a substrate hydrogen atom by the high-valent iron-oxo intermediate known as Compound I. Here, we specifically probe the influence of substrate coordination on OleT reaction partitioning through the combined use of fluorescent and electron paramagnetic resonance (EPR)-active FA probes and mutagenesis of a structurally disordered F-G loop that is distal from the heme-iron active site. Both probes are efficiently metabolized by OleT and efficiently trigger the formation of Compound I. Transient fluorescence and EPR reveal a slow product release step, mediated by the F-G loop, that limits OleT turnover. A single-amino acid change or excision of the loop reveals that this region establishes critical interactions to anchor FA substrates in place. The stabilization afforded by the F-G loop is essential for regulating regiospecific C-H abstraction and allowing for efficient decarboxylation to occur. These results highlight a regulatory strategy whereby the fate of activated oxygen species can be controlled at distances far removed from the site of chemistry.
Keyphrases
- hydrogen peroxide
- transcription factor
- living cells
- amino acid
- single molecule
- fatty acid
- small molecule
- quantum dots
- nitric oxide
- minimally invasive
- fluorescence imaging
- squamous cell carcinoma
- radiation therapy
- dna methylation
- gene expression
- cancer therapy
- brain injury
- genome wide
- subarachnoid hemorrhage
- bone mineral density
- electron transfer
- structural basis
- drug delivery
- iron deficiency