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 H2O2, O2− 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 O2 on the seconds timescale in the absence of prime substrate, and uncoupled during turnover to produce H2O2; neither O2− nor OH• were detected. In contrast, FIH was unreactive toward O2 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.

Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

“…An additional benefit is that controlled O 2 activation by FIH would avoid ROS production (21), preventing anomalous oxidations. Although it has been shown that decarboxylation of αKG and hydroxylation of CTAD by FIH is tightly coupled,(14) the mechanistic strategy used by FIH to ensure that O 2 activation leads to CTAD hydroxylation with high fidelity remains unclear.…”

“…The CTAD peptide corresponding to the C-terminal activation domain of human HIF-1α, (HIF-1α 788–826 ) contained a Cys 800 → Ala point mutation(21): DESGLPQLTSYDAEV N APIQGSRNLLQGEELLRALDQVN. The CTAD peptide was purchased as a desalted peptide from EZBiolab (Carmel, IN, USA) with free N and C-termini.…”

Electron flow through biological molecules: does hole hopping protect proteins from oxidative damage?

“…The ferryl complex abstracts a hydrogen atom from the substrate and HO • rebound leads to hydroxylated product (Denisov et al, 2005; Hausinger, 2004; Whitehouse et al, 2012). For enzymes with broad substrate specificities, or when operating in the presence of xenobiotic compounds, the fidelity of substrate oxidation is less than 100%, with potentially damaging consequences (Chen et al, 2008; De Matteis et al, 2012; Denisov et al, 2007a; Grinkova et al, 2013; Saban, Flagg & Knapp, 2011; Staudt, Lichtenb. F & Ullrich, 1974).…”

Oxygen sensing strategies in mammals and bacteria

“…The mechanical linkage between HIF-1α binding and O 2 -activation remain unclear in FIH, however this is central to O 2 sensing. Although FIH will inactivate in vitro through an auto-oxidation reaction in the absence of HIF-1α [68, 72], HIF-1α binding stimulates the O 2 reactivity of FIH many-fold. MCD and CD data of FIH in solution revealed that the Fe(II) cofactor geometry shifts from 6-coordinate to a mixture of 5/6-coordinate upon HIF-1α binding [67], in agreement with the X-ray crystal structure of HIF-1α bound FIH [73] and suggesting that aquo release may limit the O 2 reactivity [74].…”