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

Samir F El-Mashtoly,Satoru Nakashima,Atsunari Tanaka,Toru Shimizu,Teizo Kitagawa

doi:10.1074/jbc.m801262200

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 O(2), 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 non-hydrogen-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.

Highlights

The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity
The crystal structure of the O2-bound form of Ec DOSH showed that Arg-97 forms hydrogen bonds with the heme-coordinated O2 (Fig. 1B), while the interactions of the hemebound CO or NO with the surrounding residues are not known
Interactions between Heme-bound Ligand and Distal Residues—In the present study, we have investigated the interactions of heme-bound CO and NO with the surrounding residues by monitoring the RR spectra for several mutants (E93I, M95A, R97I, and F113L)

Summary

Introduction

The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. It is believed that the structural changes in the heme vicinity caused by ligand binding to the heme provide the initial event in the gas sensing, followed by intramolecular signal transduction from the heme to the functional domain, and regulating the PDE activity. In the reduced form (Fig. 1A), Met-95 and His-77 are the heme axial ligands in the distal and proximal sides, respectively, and Met-95 forms a hydrogen bond with heme 7-propionate. Upon O2 binding to the reduced heme, Met-95 is replaced by O2 and heme 7-propionate forms a hydrogen bond with Arg-97 instead of Met-95 This stabilizes the heme-coordinated O2 (Fig. 1B). The replacement of a distal axial ligand from Met-95 to O2 perturbs the heme 7-propionate hydrogen bonding network, resulting in large conformational changes in the FG loop [10]. The crystal structure provides information about the O2-free (reduced) and O2-bound forms [10], there is not nym formed from the names of proteins in which imperfect repeat sequences were initially recognized (the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM)); TR3, time-resolved resonance Raman; WT, wild type; Mb, myoglobin; 6c-ls, six-coordinate low spin state; 5c-hs, 5-coordinate high spin state; RR, resonance Raman

Results

Discussion

Conclusion