Exploring photocatalysis in Ga-PPIX-reconstituted dehaloperoxidase B

We reconstituted native dehaloperoxidase B (DHP B) with Ga protoporphyrin IX (PPIX) to create a light-driven enzyme capable of photocatalytic substrate oxidation, and explored the variant's ability to photooxidize substrates known to bind internally to the native enzyme. Mechanistic probes, as well as time- and light-intensity-dependent studies, revealed that Ga-DHP B catalyzes substrate oxidation via singlet oxygen ( 1 O 2 ) generation under visible (400-700 nm) light illumination. High-performance liquid chromatography (HPLC) and gas chromatography (GC) were used to quantify the percentage of substrate loss and product formation, and an in-depth study of 5-bromoindole (5BI) photocatalysis by Ga-DHP B revealed product distributions that were different between the typical H 2 O 2 -driven chemistry for WT DHP and the new photocatalytic singlet oxygen mechanism revealed here. The results show that DHP, as a globin with a unique active site pocket, is a photoenzyme whose chemical reactivity is highly dependent on the unique internal binding mode of substrates within the distal cavity, and whose photooxidation via 1 O 2 is likely mediated by the distal histidine that is typically involved in substrate stabilization and/or the activation of H 2 O 2 . This study strengthens the hypothesis that the mode of internal substrate binding correlates with enzyme reactivity in DHP, regardless of H 2 O 2 -initiated or photoinduced oxidation.