Abstract
Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant approximately 1 mum), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O(2)) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated approximately 10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.
Highlights
36146 JOURNAL OF BIOLOGICAL CHEMISTRY binding and release of external ligands in an associated hemebinding sensor domain (1–3)
Gel filtration is useful for estimating equilibrium constants between protein subunits, it is considerably less accurate for determining the absolute Mr and aggregation state of a protein, unless the Mr markers belong to the same protein family as the protein of interest
For this reason we used in parallel the static light scattering technique (MALLS), which allows the measuring of absolute molar masses and sizes of molecules without having to rely on the calibration of standards and assumptions of their conformation
Summary
Expression and Purification of EcDos and EcDosH—The gene coding for the full-length protein EcDos (corresponding to codons 1–799) was amplified by PCR using the primers 5Ј-atg aag cta acc gat gcg gat-3Ј (forward) and 5Ј-tca gat ttt cag cgg taa cac-3Ј (reverse) and subsequently cloned into a pBAD TOPO TA cloning vector (Invitrogen). Because of the low pI of EcDosH, the samples were loaded on the column equilibrated with 50 mM Tris/ HCl buffer, pH 8.5, and the protein was eluted at 50 mM NaCl. The concentrated material was loaded on a SuperoseTM 12 HR 16/50 (GE Healthcare) column equilibrated with PBS, pH 7.5. Pure ferric EcDos, EcDosH, and FixLH were found to have absorption ratios (A280 nm/AmaxSoret) of 1, 0.36, and 0.24, respectively, very similar to those published previously (1, 13, 24) These comparisons, as well as the comparison with predicted spectra based on protein composition (not shown), indicate that the heme to monomer ratio was close to 1 for all of our preparations. For this reason we used an enzymatic system with ferredoxin as the terminal electron donor (26), achieving full reduction of the oxidized proteins within 1 min
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