The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri

Figure 6

AhbD from M. barkeri acts as heme synthase in vitro. (a) UV-visible absorption spectrum of purified AhbD after in vitro iron-sulfur cluster reconstitution. (b) HPLC analysis of the tetrapyrrole content of AhbD enzyme activity assay mixtures. The in vitro activity assay mixture contained reconstituted AhbD (5 μM), Fe-COPRO III (20 μM), SAM (500 μM), and sodium dithionite (1 mM) as reducing agent and was incubated at 17°C. The tetrapyrrole content of the mixture was analyzed by HPLC after 0 h (upper panel), 6 h (second panel), and 15 h (third panel) of incubation. After 6 h the formation of heme was detected (37.5 min) as well as the formation of a potential monovinyl intermediate (30.1 min). After 15 h of incubation the substrate Fe-COPRO III was completely consumed as well as the potential reaction intermediate and heme was detected as the sole reaction product. In contrast, no heme formation was observed after 15 h in a control reaction in which the purified AhbD was omitted from the assay mixture.