Structural and Vibrational Characterization of the Chromophore Binding Site of Bacterial Phytochrome Agp1.

Agp1 is a prototypical bacterial phytochrome from Agrobacterium fabrum harboring a biliverdin cofactor which reversibly photoconverts between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) states. The reaction mechanism involves the isomerization of the bilin-chromophore followed by large structural changes of the protein matrix that are coupled to protonation dynamics at the chromophore binding site. Histidines His250 and His280 participate in this process. Although the three-dimensional structure of Agp1 has been solved at high resolution, the precise position of hydrogen atoms and protonation pattern in the chromophore binding pocket has not been investigated yet. Here, we present protonated structure models of Agp1 in the Pr state involving appropriately placed hydrogen atoms that were generated by hybrid quantum mechanics/molecular mechanics- and electrostatic calculations and validated against experimental structural- and spectroscopic data. Although the effect of histidine protonation on the vibrational spectra is weak, our results favor charge neutral H250 and H280 both protonated at Nε. However, a neutral H250 with a proton at Nε and a cationic H280 may also be possible. Furthermore, the present QM/MM calculations of IR and Raman spectra of Agp1 containing isotope-labeled BV provide a detailed vibrational assignment of the biliverdin modes in the fingerprint region.

Protonation-Dependent Structural Heterogeneity in the Chromophore Binding Site of Cyanobacterial Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Photoisomerization of the linear methine-bridged tetrapyrrole triggers transient proton translocation events in the chromophore binding pocket (CBP) leading to major conformational changes of the protein matrix that are in turn associated with signaling. By combining pH-dependent resonance Raman and UV-visible absorption spectroscopy, we analyzed protonation-dependent equilibria in the CBP of Cph1 involving the proposed Pr-I and Pr-II substates that prevail below and above pH 7.5, respectively. The protonation pattern and vibrational properties of these states were further characterized by means of hybrid quantum mechanics/molecular mechanics calculations. From this combined experimental-theoretical study, we were able to identify His260 as the key residue controlling pH-dependent equilibria. This residue is not only responsible for the conformational heterogeneity of CBP in the Pr state of prokaryotic phytochromes, discussed extensively in the past, but it constitutes the sink and source of protons in the proton release/uptake mechanism involving the tetrapyrrole chromophore which finally leads to the formation of the Pfr state. Thus, this work provides valuable information that may guide further experiments toward the understanding of the specific role of protons in controlling structure and function of phytochromes in general.