H2 generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water
when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H2 from water and sunlight using the
cells’ endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-
hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but
are very O2 sensitive. Certain filamentous cyanobacteria protect nitrogenase from O2 by sequestering the enzyme within internally
micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella
oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter PhetN. Active [FeFe]-hydrogenase
was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism
was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected
the [FeFe]-hydrogenase against inactivation by O2.