Vibrational coherence spectroscopy of the heme domain in the CO-sensing transcriptional activator CooA

Abstract

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 ν(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal α-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 ν(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.

Figure 1

The crystal structure of imidazole bound ferrous Ch-CooA obtained from PDB: 2FMY. The Ch-CooA consists of two symmetric A and B monomers, colored red and blue, respectively. Each monomer consists of the lightly colored N-terminal heme domains and the C-terminal DNA-binding domains are colored in normal tones. The recognition F-helix is shown in a darker color. The heme and imidazole are shown in green and yellow respectively. The coordination structures of Rr-CooA and Ch-CooA are shown in the lower panel for the ferric, ferrous and CO bound forms.

Figure 2

The upper panel compares the electronic absorption spectra of the ferric, ferrous and ferrous CO-bound forms of Ch-CooA at pH 8. The Soret absorption maxima are located at 415, 424 and 421 nm for the respective states. The lower panel displays the normalized time resolved transmittance (ΔT) of the above complexes obtained with pump/probe excitation at 420 nm for ferric and 435 nm for the ferrous and CO bound complexes. The ferric transient response shows a bleaching recovery signal (ΔT > 0). In contrast, the ferrous and CO bound forms show a strong transient absorption signal (ΔT < 0) at 435 nm that indicates ligand photolysis in both cases. The time constants are reported in the text.

Figure 3

The correlation between the Raman and coherence spectra for ferric Ch-CooA. The Raman spectrum (upper red) was measured with excitation at 413.1 nm, whereas the open band (lower green) and detuned (middle blue) coherence spectra were measured at a carrier wavelength of 420 nm. The detuned coherence data were collected with a 0.5 nm spectral window, located 5 nm to the blue of the carrier wavelength (415 nm). The insets show time domain oscillation data (circles) and LPSVD fits (lines) corresponding to the open band (green) and detuned (blue) detection methods. There is a very good correlation between the Raman and coherence spectral frequencies within accuracy of ± 5 cm^-1

Figure 4

The open-band and detuned coherence spectra of ferrous Ch-CooA at pH 8. The pump and probe wavelength are given in the inset. The left panels show the oscillatory components (circles) and LPSVD fits (solid red lines). The LPSVD components corresponding to the dominant mode are also shown (blue solid lines), along with the extracted phases. The right panels show the corresponding power spectrum amplitudes. The correlated modes are shown by dotted lines.

Figure 5

The femtosecond coherence signal of CO bound Ch-CooA obtained using detuned detection. The pump wavelength is 435 nm and the probe is at 429 nm (detuned 6 nm to the blue of the carrier wavelength). The inset shows the signal from 120 fs to 2.5 ps. The oscillatory components of the signal are clearly apparent in the inset. They correspond to the strongly coupled coherent nuclear motion following CO photolysis.

Figure 6

The open-band and detuned coherence spectra of the CO bound form of Ch-CooA at pH 8. The pump and probe wavelengths are given in the inset. The left panel shows the oscillatory components (circles) and the LPSVD fits (solid red lines). The LPSVD components corresponding to the dominant mode are also shown (blue solid lines), along with the extracted phases. The right panels show the corresponding power spectrum amplitudes. The correlated modes are shown by dotted lines.

Figure 7

The open-band and detuned coherence spectra of Ch-CooA-CO that has bound to specific DNA at pH 8. The components of the figure are analogous to Fig.6.

Figure 8

The crystal structures and NSD analysis of the heme in imidazole-bound ferrous Ch-CooA and the CO bound form of LL-Ch-CooA. The displacement along each of the low frequency normal mode unit vectors of Fe porphine is given in mass weighted coordinates (amu^1/2Å). The color coding for the modes is propellering (blue), ruffling (green), saddling (red), waving-x (light blue), waving-y (brown), doming (purple), and inverse doming (gray). The crystal structures are extracted from the protein data bank 2FMY and 2HKX for imidazole-bound ferrous Ch-CooA and CO bound LL-Ch-CooA, respectively. The NSD analysis of ferrous Ch-CooA shows strong ruffling and saddling distortions, whereas the CO bound form shows the ruffling distortion is maintained but the saddling distortion is strongly diminished.

Figure 9

The detuned coherence spectra of ferrous, and Ch-CooA-CO with and without DNA bound. The pump and probe wavelengths are 435 and 429 nm respectively for all the three forms. The correlated modes are shown by dotted lines. The spectra of Ch-CooA-CO with and without bound DNA are similar and indicate that no significant difference in heme distortion when DNA is bound. This suggests that kinetic effects (to be published) are likely associated with changes in the distal pocket environment when DNA binds.