Fig. 2
Spectroscopic studies showing formation of a catalytically inactive PHD2.Fe(III).2OG complex with a blue chromophore. (a) UV-vis spectra of an O2 exposed mixture of PHD2 (300 µM), (NH4)2Fe(II)(SO4) (250 µM), 2OG (3 mM) and HIF2α-CODD (250 µM). Grey-scale plots show 1 min intervals up to 10 min. Spectra between 10 and 30 min are in 5 min intervals. (b) UV-vis spectra of anaerobic and an O2 exposed mixture of PHD2 (300 µM), (NH4)2Fe(II)(SO4) (250 µM), 2OG (3 mM), HIF2α-CODD (250 µM if present). For spectra 3 and 4, the samples were exposed to O2 for 60 min. (c) The extent of hydroxylation when a PHD2.Fe(II).2OG mixture is added to an HIF1α-CODD556 − 574 solution, with or without prior O2 exposure of the PHD2.Fe(II).2OG mixture. A clear decrease in hydroxylation is observed for the O2 exposed mixture after ~ 30 and 60 min. No activity loss was observed for the anaerobic control. The results are means of 3 independent assays (n = 3; mean ± SD). (d) Comparison of hydroxylation observed when a PHD2.Fe(II) mixture is added to HIF1α-CODD556 − 574 and 2OG, both with and without prior O2 exposure of the PHD2.Fe(II) mixture. No activity loss was observed for the anaerobic control and the O2 exposed sample. The results are means of 3 independent runs (n = 3; mean ± SD). (e) EPR spectra of anaerobic PHD2.Fe(II).2OG (orange line) and O2 exposed (blue line, 60 min) PHD2.Fe(II).2OG. (f) EPR spectra of anaerobic PHD2.Fe(II) (orange line) and O2 exposed (blue line, 60 min) PHD2.Fe(II).
