Escherichia coli when growing anaerobically synthesizes three [NiFe]-hydrogenases. We investigated the consequences on hydrogenase levels, enzyme activity and gene expression of deleting the ferric iron-uptake regulator Fur, which is required to coordinate intracellular iron levels. Total hydrogenase activity was reduced between 80 and 90% in the fur mutant. Hydrogen production by the formate hydrogenlyase pathway was strongly reduced. Analysis of lacZ fusions to the various hydrogenase structural operons demonstrated that regulation of hya and hyb was not transcriptional, while the effect of the fur mutation on hyc was partly due to reduced intracellular formate. Immunological analysis of the hydrogenase large subunits revealed that the absolute levels of the enzymes were reduced suggesting that either post-transcriptional or post-translational control, possibly through enhanced enzyme turnover, was a major cause of reduced activity. A mutant defective in multiple iron transport systems also essentially lacked hydrogenase activity highlighting the importance of intracellular iron availability in regulating hydrogenase synthesis. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.