Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen

The prolyl hydroxylase domain proteins (PHDs) catalyse the post-translational hydroxylation of the hypoxia-inducible factor (HIF), a modification that regulates the hypoxic response in humans. The PHDs are Fe(II)/2-oxoglutarate (2OG) oxygenases; their catalysis is proposed to provide a link between cellular HIF levels and changes in O-2 availability. Transient kinetic studies have shown that purified PHD2 reacts slowly with O-2 compared with some other studied 2OG oxygenases, a property which may be related to its hypoxia-sensing role. PHD2 forms a stable complex with Fe(II) and 2OG; crystallographic and kinetic analyses indicate that an Fe(II)-co-ordinated water molecule, which must be displaced before O-2 binding, is relatively stable in the active site of PHD2. We used active site substitutions to investigate whether these properties are related to the slow reaction of PHD2 with O-2. While disruption of 2OG binding in a R383K variant did not accelerate O-2 activation, we found that substitution of the Fe(ll)-binding aspartate for a glutamate residue (D315E) manifested significantly reduced Fe(II) binding, yet maintained catalytic activity with a 5-fold faster reaction with O-2. The results inform on how the precise active site environment of oxygenases can affect rates of O-2 activation and provide insights into limiting steps in PHD catalysis.