Abstract
Gas sensory heme proteins respond to their environment by binding a specific gas molecule to heme and transmitting this primary binding signal to the protein. How the binding signal is transmitted from the heme to the protein remains to be clarified. Using UV resonance Raman (UVRR) spectroscopy, we investigated this pathway in sperm whale myoglobin as a model gas sensory heme protein. Based on the UVRR data and the effects of deleting one of three important pathways (His-93, 6-propionate, or 7-propionate), we determined the changes in the conformation of globin that occur upon binding of CO, nitric oxide (NO), or O(2) to heme and how they are transmitted from heme to globin. The UVRR results show that heme discriminates different ligands, resulting in different conformations in the globin protein. Specifically, NO induces changes in the spectrum of Trp residues in the A-helix that are significantly different from those induced by O(2) or CO binding. On the other hand, binding of O(2) to heme produces changes in the Tyr residues of the H-helix that are different from those induced by CO or NO binding. Furthermore, we found that cleavage of the Fe-His-93 covalent bond eliminates communication to the terminal region of the H-helix and that the 7-propionate hydrogen-bonding network is essential for transmitting the CO or NO binding signal to the N and C termini. Finally, the 6-propionate is important only for NO binding. Thus, the hydrogen-bonding network in the protein appears to be critical for intramolecular signal transduction in gas sensory heme proteins.
Highlights
Protein that can reversibly bind diatomic gaseous molecules (O2, nitric oxide (NO),2 and CO)
We examined sperm whale Mb (swMb) by ultraviolet resonance Raman (UVRR) spectroscopy to explore the role of hydrogen bonds in this communication process
Because of the importance of the physiological ligand, O2, for Mbs, we examined the UVRR spectral changes of NT Mb upon binding of O2
Summary
Thermore, we monitored the UVRR spectral changes of Trp and Tyr residues that occur upon CO and NO binding to versions of Mb in which the predicted pathways for transmission of conformational changes (His-93, 6-propionate, or 7-propionate) were eliminated by mutation or chemical modification of the heme. As an internal intensity standard for UVRR difference spectra, 400 and 100 mM Na2SO4 were included in the samples for excitation at 229 and 244 nm, respectively. The O2-bound form was prepared by passing the dithionite-reduced Mb protein through a small Sephadex G25 column under aerobic conditions and saturating the sample with oxygen by flushing it with pure oxygen gas in a sealed Raman cell. The laser power at the sample point was 0.2– 0.3 milliwatt, and the spectral resolutions were set at 6.9 and 7.8 cmϪ1 for 244 and 229 nm excitations, respectively. The protein sample was replaced with fresh one every 5–10 min, and the total exposure time to obtain each spectrum was ϳ1 h. The integrity of the sample after exposure to UV laser light was confirmed by comparing the visible absorption spectra obtained before and after the UVRR measurements. Raman shifts were calibrated with cyclohexane, trichloroethylene, 1,2-dichloroethane, and toluene
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