Unexpected NO-dependent DNA binding by the CooA homolog from Carboxydothermus hydrogenoformans

Abstract

CooA, the CO-sensing heme protein from Rhodospirillum rubrum, regulates the expression of genes that encode a CO-oxidation system, allowing R. rubrum to use CO as a sole energy source. To better understand the gas-sensing regulation mechanism used by R. rubrum CooA and its homologs in other organisms, we characterized spectroscopically and functionally the Fe(II), Fe(II)-NO, and Fe(II)-CO forms of CooA from Carboxydothermus hydrogenoformans. Surprisingly, and unlike R. rubrum CooA, C. hydrogenoformans CooA binds NO to form a six-coordinate Fe(II)-NO heme that is active for DNA binding in vitro and in vivo. In contrast, R. rubrum CooA, which is exquisitely specific for CO, forms a five-coordinate Fe(II)-NO adduct that is inactive for DNA binding. Based on analyses of protein variants and temperature studies, NO-dependent DNA binding by C. hydrogenoformans CooA is proposed to result from a greater apparent stability of the six-coordinate Fe(II)-NO adduct at room temperature. Results from the present study strengthen the proposal that CO specificity in the CooA activation mechanism is based on the requirement for a small, neutral distal ligand, which in turn affects the relative positioning of the ligand-bound heme.

Fig. 1.

Ch CooA binds to a P_cooF DNA fragment when exposed to either NO (filled triangles) or CO (open circles) gases. In vitro DNA-binding assays were performed by incubating a Texas red-labeled P_cooF fragment with different levels of NO- or CO-bound Fe(II) Ch CooA and measuring resulting changes in fluorescence anisotropy.

Fig. 2.

Only Ch cooA-containing E. coli strains show β-galactosidase activity when treated with the NO precursor, NaNO₃. Plates were spotted with 15 μl (A) or 45 μl (B) of 5 M NaNO₃ at the position indicated in A. Streak 1, -cooA control; streak 2, WT Ch CooA; streak 3, WT Rr CooA; streak 4, a shortened Ch CooA protein where the first four residues have been removed; streak 5, K119A/K126A Ch CooA.

Fig. 3.

NO binding to Ch CooA yields a six-coordinate Fe(II)-NO heme. Room temperature electronic absorption spectra of 10 μM Fe(II)-NO WT Ch CooA (solid line) and WT Rr CooA (dashed line) samples are shown.

Fig. 4.

The EPR spectrum of Fe(II)-NO WT Ch CooA reveals that the ligand trans to NO is a nitrogen donor. The spectrum of the ≈130 μM sample shown represents the average of 8 scans and was recorded at 10 K with a ≈9.36-GHz microwave frequency, 100-kHz modulation frequency, 2.1-G modulation amplitude, 1.6 × 10^–1 mW of microwave power, and a 6.3 × 10⁵ receiver gain.

Fig. 5.

The coordination number of the Fe(II)-NO Ch CooA heme varies with temperature. Electronic absorption spectra of a 10 μM Fe(II)-NO Ch CooA sample recorded at several temperatures from 25°C to ≈70°C are shown.