passages.id: PMID-18805587__text
passages.type: abstract
passages.text: Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor. 
  Hypoxia sensing is the generic term for pO2-sensing in humans and other higher organisms. These cellular responses to pO2 are largely controlled by enzymes that belong to the Fe(II) alpha-ketoglutarate (alphaKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O2-activation to the hydroxylation of the hypoxia inducible factor alpha (HIFalpha). Uncoupled O2-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (alphaKG + Fe)FIH-1, to air in the absence of HIFalpha. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (lambda(max) = 583 nm) arising from enzyme auto-hydroxylation of Trp296, forming an Fe(III)-O-Trp296 chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met275. The kinetics of purple FIH-1 formation were independent of Fe(II) and alphaKG concentrations, however, product yield was saturable with increasing [alphaKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate+Fe)FIH-1, or by glycerol addition to (alphaKG+Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (alphaKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O2-activation by loss of an H2O ligand from the metal center.
  