Identification of the Deprotonated Pyrrole Nitrogen of the Bilin-Based Photoreceptor by Raman Spectroscopy with an Advanced Computational Analysis.

Phytochrome and cyanobacteriochrome utilize a linear methine-bridged tetrapyrrole (bilin) to control numerous biological processes. They show a reversible photoconversion between two spectrally distinct states. This photocycle is initiated by a C═C double-bond photoisomerization of the bilin followed by its thermal relaxations with transient and/or stationary changes in the protonation state of the pyrrole moiety. However, it has never been identified which of the four pyrrole nitrogen atoms is deprotonated. Here, we report a resonance Raman spectroscopic study on cyanobacteriochrome RcaE, which has been proposed to contain a deprotonated bilin for its green-absorbing 15 Z state. The observed Raman spectra were well reproduced by a simulated structure whose bilin B ring is deprotonated, with the aid of molecular dynamics and quantum mechanics/molecular mechanics calculations. The results revealed that the deprotonation of B and C rings has the distinct effect on the overall bilin structure, which will be relevant to the color tuning and photoconversion mechanisms of the phytochrome superfamily. Furthermore, this study documents the ability of vibrational spectroscopy combined with the advanced spectral analysis to visualize a proton of a cofactor molecule embedded in a protein moiety.

Hydrogen Bonding Environments in the Photocycle Process around the Flavin Chromophore of the AppA-BLUF domain.

Three kinds of photochemical reactions are known in flavins as chromophores of photosensor proteins, reflecting the various catalytic reactions of the flavin in flavoenzymes. Sensor of blue light using the flavin FAD (BLUF) domains exhibit a unique photoreaction compared with other flavin-binding photoreceptors in that the chromophore does not change its chemical structure between unphotolyzed and intermediate states. Rather, the hydrogen bonding environment is altered, whereby the conserved Gln and Tyr residues near FAD play a crucial role. One proposal for this behavior is that the conserved Gln changes its chemical structure from a keto to an enol. We applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to AppA-BLUF. The spectra of AppA-BLUF exhibited a different feature upon 15N-Gln labeling compared with the previously reported spectra from BlrB, a different BLUF domain. The FTIR signals were interpreted from quantum mechanics/molecular mechanics (QM/MM) calculation as the keto-enol tautomerization and rotation of the Gln63 side chain in the AppA-BLUF domain. The former was consistent with the result from BlrB, but the latter was not uniquely determined by the previous study. QM/MM calculation also indicated that the infrared signal shape is influenced depending on whether a Trp side chain forms a hydrogen bond with the Gln side chain. FTIR spectra and QM/MM simulations concluded that Trp104 does not flip out but is maintained in the intermediate state. In contrast, our data revealed that the Trp residue at the corresponding position in BlrB faces outward in both states.

The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c' from the thermophilic purple sulfur bacterium Thermochromatium tepidum.

A soluble cytochrome (Cyt) c' from thermophilic purple sulfur photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c' and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c' by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c'. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c', indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c'. The control Cyt c' and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c' exhibited characteristic bands at 1,635 and 1,625 cm(-1), ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c'.