A recombinant protein overproduction system was developed in Methanosarcina acetiVorans to facilitate biochemical characterization of oxygen-sensitive metal-loenzymes from strictly anaerobic species in the Archaea domain. The system was used to overproduce the archetype of the independently evolved γ-class carbonic anhydrase. The overproduced enzyme was oxygen sensitive and had full incorporation of iron instead of zinc observed when overproduced in Escherichia coli. This, the first report of in vivo iron incorporation for any carbonic anhydrase, supports the need to reevaluate the role of iron in all classes of carbonic anhydrases derived from anaerobic environments.
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