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.
Chen
Yuan-Han
YH
Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
Comeaux
Lindsay M
LM
Herbst
Robert W
RW
Saban
Evren
E
Kennedy
David C
DC
Maroney
Michael J
MJ
Knapp
Michael J
MJ
D000693:Anaerobiosis; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007501:Iron / Q000378:metabolism; D006899:Mixed Function Oxygenases; D010100:Oxygen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D005296:Ferrous Compounds; D007501:Iron; D006899:Mixed Function Oxygenases; D010100:Oxygen; D011994:Recombinant Proteins; D012097:Repressor Proteins; C439790:HIF1AN protein, human