| Summary: | <p>Human hypoxia inducible factor (HIF) is responsible for mediating the body’s response to low O<sub>2</sub> availability. The levels and activity of HIF are regulated by four Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases, prolyl-hydroxylase domains 1-3 (PHD 1-3) and factor-inhibiting HIF (FIH), collectively termed the HIF hydroxylases. The PHDs catalyse hydroxylation of specific prolyl residues in the N - and C - terminal oxygen-dependent degradation domains of HIF-α, targeting it for degradation by the proteasome. FIH catalyses hydroxylation of an asparaginyl residue in the C-terminal transactivation domain, preventing the interaction of HIF-α with the co-transcriptional activator p300. Under hypoxic conditions, the activity of the oxygen-dependent HIF hydroxylases is reduced, causing an increase in cellular HIF-α levels/activity and triggering the transcription of genes that enable the cellular and physiological hypoxic response. </p> <p>PHD2 is reported to be the key O<sub>2</sub> sensor regulating the hypoxic response. It has also been reported to have a high <em>K</em><sub><em>m</em></sub>(O<sub>2</sub>) value and a slow reaction with O<sub>2</sub> in pre-steady state studies, kinetic features that are proposed to be related to its role as an O<sub>2</sub>-sensor. We are interested in the molecular features that enable this O<sub>2</sub>-sensing role, when other Fe(II)/2OG oxygenases react &Tilde;100-fold more rapidly with O<sub>2</sub>. To investigate whether restricted O<sub>2</sub> passage to the active site may be responsible for its unusual kinetics, our collaborators (C. Jorgensen and C. Domene, KCL) have conducted molecular dynamic studies to investigate the route of O<sub>2</sub> entry to the PHD2 active site. These studies (unpublished) have indicated that O<sub>2</sub> enters PHD2 via the interface between HIF substrate and a flexible, substrate-binding loop (the β2β3 loop) in PHD2. O2 is then proposed to reside in a stable 'E-cluster' in PHD2, before moving to the active site. This report describes work conducted to experimentally verify this predicted O<sub>2</sub> entry pathway into PHD2, and to determine whether aspects of this entry pathway contribute to the slow kinetics of the reaction of PHD2 with O<sub>2</sub>.</p> <p>To validate the role of the β2β3 loop in the reaction of PHD2 with O<sub>2</sub>, kinetic studies of PHD2 loop variants were undertaken. These variants were previously developed by Flashman <em>et al</em> such that the β2β3 loop of PHD2 was altered to include the loop sequences of PHD1 and PHD3. These loop variants demonstrated reduced O<sub>2</sub> sensitivity, and with the β2β3 loop of PHD3, a more rapid reaction with O<sub>2</sub>. These data suggest the β2β3 loop of PHD2 does indeed play an important role in O<sub>2</sub> uptake.</p> <p>Experimental validation of the stable E-cluster was then undertaken. The site directed mutagenesis of methionine 299 in PHD2 to histidine did not yield different kinetics with respect to O<sub>2</sub>, suggesting either replacement of methionine 299 with histidine does not alter the O<sub>2</sub> 'stabilising' characteristics of the E-cluster or the E-cluster is not significant in O<sub>2</sub> kinetics. Tryptophans 258 and 389 also form part of the E cluster. Laser-induced excitation of molecular O<sub>2</sub> to O<sub>2</sub>&ast; was used to attempt to modify and thus identify amino acid residues in regions where O2 was stably bound, i.e. the E-cluster. This approach was technically challenging, and results obtained were inconclusive. Further investigations using this method would require substantial optimisation.</p> <p>Stopped-flow tryptophan fluorescence quenching studies confirmed that these residues encounter a quenching species upon introduction of O<sub>2</sub>, supporting the hypothesis that they are present on the O2 uptake pathway. Interestingly, the rate of tryptophan fluorescence quenching of the PHD2<sub>variant</sub>.CODD.Fe(II).2OG complex by O<sub>2</sub> correlates with the PHD2 WT rate of product turnover suggesting the quenching is correlated with product turnover. The results reported here imply that the rate limiting step with respect to PHD2 and O<sub>2</sub> is prior to O<sub>2</sub> reaching the E cluster.</p> <p>Overall the thesis supports the hypothesis that PHD2's β2β3 loop is involved in PHD2's O<sub>2</sub>–sensing capability. Experiments were unable to verify the existence of an E-cluster, including the finding that O<sub>2</sub> encounters two tryptophans present along the proposed O2 uptake pathway in PHD2 at the same rate as PHD2 catalysis. The findings of this study suggest that the β2β3 loop of PHD2 may be a key molecular feature involved in O2 sensing by PHD2, a factor that could be taken into account when designing PHD2 inhibiting or activating therapies. </p>
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