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Functionally critical elements of CooA-related CO sensors.

ABSTRACT: CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in Escherichia coli, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from Azotobacter vinelandii and Carboxydothermus hydrogenoformans corroborated the in vivo data by revealing the ability of CO to bind to these hemoproteins and stimulate their binding at specific DNA sequences. These data, as well as the patterns of conserved residues in the homologs, are compared to what is already known about functionally important residues in the CooA protein of R. rubrum. The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.

SUBMITTER: Youn H

PROVIDER: S-EPMC344431 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Functionally critical elements of CooA-related CO sensors.

Youn Hwan H Kerby Robert L RL Conrad Mary M Roberts Gary P GP

Journal of bacteriology 20040301 5

CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in Escherichia coli, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from Azotobacter vinelandii and Carboxydothermus hydrogenoformans corroborated the in vivo data by revealing the ability of CO to bind to t ...[more]

PMID: 14973040

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Project description:Femtosecond vibrational coherence spectroscopy was used to investigate the low-frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (k(B)T = 200 cm(-1)) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods. Here, we present the low frequency coherence spectra of the ferric, ferrous, and CO-bound forms of Ch-CooA in order to compare the protein-induced heme distortions in its active and inactive states. Distortions take place predominantly along the coordinates of low-frequency modes because of their weak force constants, and such distortions are reflected in the intensity of the vibrational coherence signals. A strong mode near ~90 cm(-1) in the ferrous form of Ch-CooA is suggested to contain a large component of heme ruffling, consistent with the imidazole-bound ferrous heme crystal structure, which shows a significant protein-induced heme distortion along this coordinate. A mode observed at ~228 cm(-1) in the six-coordinate ferrous state is proposed to be the ?(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal ?-amino axial ligand takes place. This is followed by a rapid (~8.5 ps) transient absorption recovery, analogous to methionine rebinding in photolyzed ferrous cytochrome c. We have also studied CO photolysis in CooA, which revealed very strong photoproduct state coherent oscillations. The observation of heme-CO photoproduct oscillations is unusual because most other heme systems have CO rebinding kinetics that are too slow to make the measurement possible. The low frequency coherence spectrum of the CO-bound form of Ch-CooA shows a strong vibration at ~230 cm(-1) that is broadened and up-shifted compared to the ?(Fe-His) of Rr-CooA (216 cm(-1)). We propose that the stronger Fe-His bond is related to the enhanced thermal stability of Ch-CooA and that there is a smaller (time dependent) tilt of the histidine ring with respect to the heme plane in Ch-CooA. The appearance of strong modes at ~48 cm(-1) in both the ferrous and CO-bound forms of Ch-CooA is consistent with coupling of the heme doming distortion to the photolysis reaction in both samples. Upon CO binding and protein activation, a heme mode near 112 ± 5 cm(-1) disappears, probably indicating a decreased heme saddling distortion. This reflects changes in the heme environment and geometry that must be associated with the conformational transition activating the DNA-binding domain. Protein-specific DNA binding to the CO-bound form of Ch-CooA was also investigated, and although the CO rebinding kinetics are significantly perturbed, there are negligible changes in the low-frequency vibrational spectrum of the heme.

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Functionally critical elements of CooA-related CO sensors.

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Functionally critical elements of CooA-related CO sensors.

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