title: Uncoupled O2-activation in the human HIF-asparaginyl hydroxylase, FIH, does not produce reactive oxygen species.
abstract: The factor inhibiting HIF (FIH) is one of the primary oxygen sensors in human cells, controlling gene expression by hydroxylating the α-subunit of the hypoxia inducible transcription factor (HIF). As FIH is an alpha-ketoglutarate dependent non-heme iron dioxygenase, oxygen activation is thought to precede substrate hydroxylation. The coupling between oxygen activation and substrate hydroxylation was hypothesized to be very tight, in order for FIH to fulfill its function as a regulatory enzyme. Coupling was investigated by looking for reactive oxygen species production during turnover. We used alkylsulfatase (AtsK), a metabolic bacterial enzyme with a related mechanism and similar turnover frequency, for comparison, and tested both FIH and AtsK for H(2)O(2), O(2)(-) and OH formation under steady and substrate-depleted conditions. Coupling ratios were determined by comparing the ratio of substrate consumed to product formed. We found that AtsK reacted with O(2) on the seconds timescale in the absence of prime substrate, and uncoupled during turnover to produce H(2)O(2); neither O(2)(-) nor OH were detected. In contrast, FIH was unreactive toward O(2) on the minutes timescale in the absence of prime substrate, and tightly coupled during steady-state turnover; we were unable to detect any reactive oxygen species produced by FIH. We also investigated the inactivation mechanisms of these enzymes and found that AtsK likely inactivated due to deoligomerizion, whereas FIH inactivated by slow autohydroxylation. Autohydroxylated FIH could not be reactivated by dithiothreitol (DTT) nor ascorbate, suggesting that autohydroxylation is likely to be irreversible under physiological conditions.
authors: Department of Chemistry, University of Massachusetts, Amherst, MA 01003, United States.
authors: Evren
authors: E
authors: Saban
authors: Shannon C
authors: SC
authors: Flagg
authors: Michael J
authors: MJ
authors: Knapp
mesh_terms: D049308:Dioxygenases / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D013429:Sulfatases / Q000737:chemistry / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C439790:HIF1AN protein, human; D013429:Sulfatases; C019244:alkylsulfatase; D010100:Oxygen