Mechanism of the CO-sensing heme protein CooA: new insights from the truncated heme domain and UVRR spectroscopy

Abstract

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.

Fig. 1

X-ray crystal structures of Rr-CooA (left, Protein Data Bank (PDB) code 1FT9) and CRP (PDB code 1G6N) showing the difference in orientation of the DNA binding domains, relative to the effector binding domains. In the CooA crystal, the C-helix connecting the effector and DNA binding domains is fully extended in the B chain but is bent over in the A chain (though not as much as in CRP), probably due to crystal forces [9].

Fig. 2

Overlay of CO-bound LL-ChCooA (orange) and CO-free RrCooA (blue), aligned via residues 29–99 in Ch- and the homologous residues 24–94 in Rr-CooA (A chain). (RMS = 1.30 Å for 71 C_α atoms). Shifts in the portions of the hemes, and of the opposite C-helix (carrying the UVRR indicator, Trp110), are indicated by pink arrows. In LL-ChCooA-CO, the Arg138-Glu59 (Arg143-Glu64) saltbridge is broken, the hinge is bent and the D-helix, which is connected to the DNA-binding domain, is reoriented. The heme displacement weakens the His77-Asn42 (His82-Asn47) H-bond. Also shown are the Tyr55-Glu83 (Tyr60-Gln88) contacts. The box is a close-up view of the heme pocket.

Fig. 3

RrCooA UVRR spectra with and without CO, and the difference spectra (CooA-CO minus CooA) for the indicated variants (5 × amplification). Band frequencies and assignments are labeled.

Fig. 4

Heme-resonant FeC and CO stretching RR bands for the indicated RrCooA-CO variants.

Fig. 5

ν_Fe-C/ν_CO back-bonding plot showing data for CO adducts of myoglobin variants (□) [6] and of the CooA variants in this (●) and previous (○, [7]) studies. The CooA variants deviate horizontally from the Mb line, as expected for weakening of the Fe-His bond. The lower and upper inserts show the proximal histidine H-bond arrangements in Mb and CooA, respectively.

Fig. 6

Time-resolved resonance Raman spectra (438-nm probe and 527-nm pump) showing the ν₄ bands obtained after photolysis of CooA-CO at the indicated delay times.

Fig. 7

Changes in the percentages of CooA-CO, CooA, and CooA (5c) as a function of time, based on deconvolution of the Raman ν₄ bands. The inset shows the band deconvolution into components (see “Methods”).

Fig. 8

View of LL-ChCooA-CO (chain A) [15], with Pro and Tyr modeled as substitutions for Leu7 and Leu127, showing the proposed H-bond docking of the N-terminal tail in the active conformation of (A/R)YLLRL variants of RrCooA, in which Pro2 and Tyr122 are the position 7 and 127 homologs.