In vitro specific activity enzyme assays

Specific activity measurements were measured spectrophotometrically using an 8453 UV-Vis spectrophotometer in kinetics mode (Agilent Technologies, Santa Clara, CA, USA). Precorrin-2 dehydrogenase activity and sirohydrochlorin cobalt chelation were monitored by an increase or decrease of sirohydrochlorin concentration at an absorbance at 376 nm, respectively. All reactions were completed in anaerobic 10 mm Quartz septa-sealed cuvettes. The precorrin-2 dehydrogenase reaction contained 2.5 µm precorrin-2, 1 mm NAD⁺, and 1–10 µg of CysG in 50T100 pH 8.0. The sirohydrochlorin cobalt chelatase reaction contained 2.5 µm sirohydrochlorin, 20 µm CoCl₂, and 1–10 µg of CysG in 50T100 pH 8.0. Reactions were started by injection of CysG enzyme and recorded concurrently with a blank to correct for any oxidation. Assays were repeated in triplicate for each of the CysG variants. Owing to the extreme air sensitivity of the substrates and the complex mixture that results from the enzyme-coupled synthesis, complete kinetic analysis was not feasible²⁶.