Roles of Arg-97 and Phe-113 in regulation of distal ligand binding to heme in the sensor domain of Ec DOS protein: Resonance raman and mutation study

Abstract

The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O2, CO, or NO to a reduced heme significantly enhances the PDE activity toward 3′,5′-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 → Ile, Met-95 → Ala, Arg-97 → Ile, Arg-97 → Ala, Arg-97 → Glu, Phe-113 → Leu, and Phe-113 → Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and nonhydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.