Pressure effects reveal that changes in the redox states of the heme iron complexes in the sensor domains of two heme-based oxygen sensor proteins, EcDOS and YddV, have profound effects on their flexibility.

The catalytic activity of a heme-based oxygen sensor phosphodiesterase from Escherichia coli (EcDOS) towards cyclic diGMP is regulated by the redox state of the heme iron complex in the enzyme's sensing domain and the association of external ligands with the iron center. Specifically, the Fe (II) complex is more active towards cyclic diGMP than the Fe(III) complex, and its activity is further enhanced by O-2 or CO binding. In order to determine how the redox state and coordination of the heme iron atom regulate the catalytic activity of EcDOS, we investigated the flexibility of its isolated N-terminal heme-binding domain (EcDOS-heme) by monitoring its spectral properties at various hydrostatic pressures. The most active form of the heme-containing domain, i.e. the Fe(II)-CO complex, was found to be the least flexible. Conversely, the oxidized Fe(III) forms of EcDOS-heme and its mutants had relatively high flexibilities, which appeared to be linked to the low catalytic activity of the corresponding intact enzymes. These findings corroborate the suggestion, made on the basis of crystallographic data, that there is an inverse relationship between the flexibility of the heme-containing domain of EcDOS and its catalytic activity. The Fe(II)-CO form of the heme domain of a second heme-based oxygen sensor, diguanylate cyclase (YddV), was also found to be quite rigid. Interestingly, the incorporation of a water molecule into the heme complex of YddV caused by mutation of the Leu65 residue reduced the flexibility of this heme domain. Conversely, mutation of the Tyr43 residue increased its flexibility.