Properties of prolyl 4-hydroxylase containing firmly-bound iron.

1. Prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) was isolated in a form containing iron (0.85-1.1 mol Fe/mol enzyme). 2. The enzyme was pure according to gel electrophoresis and had a high specific activity (1.8-2.6 mumol . mi-1 . mg-1). 3. Experiments with metal chelators showed this iron to be firmly bound and to be required for catalytic activity. 4. According to EPR spectrometry the bound iron is not part of a [2Fe-2S] or a [4Fe-4S] cluster. 5. The enzyme activity is to a large extent independent of added Fe2+. 6. The enzyme activity is completely dependent on ascorbate. 7. In the absence of ascorbate but the presence of substrates the enzyme is irreversibly inactivated. 8. Continuous measurement of enzyme activity was possible by following oxygen uptake.