Finding intermediates in the O2 activation pathways of non-heme iron oxygenases.
Intermediates in the reaction cycle of an oxygenase are usually very informative with respect to the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.
Kovaleva
E G
EG
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, Minneapolis, Minnesota 55455, USA.
Neibergall
M B
MB
Chakrabarty
S
S
Lipscomb
J D
JD
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019159:Nonheme Iron Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism
D019159:Nonheme Iron Proteins; D017382:Reactive Oxygen Species; D010105:Oxygenases; D049308:Dioxygenases; C021555:methane monooxygenase; C439587:extradiol dioxygenase; D010100:Oxygen