Misao Mizuno

Changes in the hydrogen-bond network around the chromophore of photoactive yellow protein in the ground and excited states.

Changes in the hydrogen-bond (HB) network around the chromophore, p-coumaric acid (pCA), in the ground pG and excited pG* states were investigated for wild type (WT) photoactive yellow protein (PYP) and its mutants using ultraviolet resonance Raman (UVRR) spectroscopy. The intensity depletion of Tyr UVRR bands was observed upon photoexcitation of pCA to the pG* state. The spectral change was ascribed to strengthening of HB between pCA and Tyr42. Comparison of Raman intensities indicated that, in the pG state, the HB between pCA and Tyr42 in WT is a short HB, which is weaker than that in E46Q mutant. In the pG* state, the HB network around pCA of WT is similar to that of E46Q mutant. The present results demonstrate that the HB between pCA and Tyr42 and that between pCA and Glu46 are correlated with each other in the HB network.

Direct Observation of Vibrational Energy Flow in Cytochrome c

Vibrational energy flow in ferric cytochrome c has been examined by picosecond time-resolved anti-Stokes ultraviolet resonance Raman (UVRR) measurements. By taking advantage of the extremely short nonradiative excited state lifetime of heme in the protein (<< ps), excess vibrational energy of 20000-25000 cm(-1) was optically deposited selectively at the heme site. Subsequent energy relaxation in the protein moiety was investigated by monitoring the anti-Stokes UVRR intensities of the Trp59 residue, which is a single tryptophan residue involved in the protein that is located close to the heme group. It was found from temporal changes of the anti-Stokes UVRR intensities that the energy flow from the heme to Trp59 and the energy release from Trp59 took place with the time constants of 1-3 and ~8 ps, respectively. These data are consistent with the time constants for the vibrational relaxation of the heme and heating of water reported for hemeproteins. The kinetics of the energy flow were not affected by the amount of excess energy deposited at the heme group. These results demonstrate that the present technique is a powerful tool for studying the vibrational energy flow in proteins.

Picosecond time-resolved ultraviolet resonance Raman spectroscopy of bacteriorhodopsin: primary protein response to the photoisomerization of retinal.

Protein dynamics in the primary processes during the bacteriorhodopsin (BR) photocycle under physiological conditions were investigated by measuring picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of the BR suspended solution at ambient temperature. We used a 565 nm pump pulse to initiate the BR photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, that is, Trp86, Trp182, Tyr57, and Tyr185. The 225 nm UVRR spectra exhibited bleaching of intensity for all the tryptophan bands within the instrumental response, followed by recovery with a time constant of 30 ps and no further changes up to 1 ns. This suggests that the stepwise structural changes in the tryptophan residues proceed in response to the retinal photoreaction. It is concluded that the initial intensity bleach arises from the J-intermediate formation and the 30 ps recovery is associated with the K-KL transition. The 30 ps process in the BR photocycle has been detected for the first time. In the 238 nm UVRR spectra, spectral features attributable to the K and KL intermediates were observed. The observed spectral changes showed that the temporal behaviors of the observed spectral changes in each Raman band of both tryptophan and tyrosine were different. This indicates that the spectral changes originated from structural changes of at least two tryptophan and two tyrosine residues.

Observing Vibrational Energy Flow in a Protein with the Spatial Resolution of a Single Amino Acid Residue.

One of the challenges in physical chemistry has been understanding how energy flows in a condensed phase from the microscopic viewpoint. To address this, space-resolved information at the molecular scale is required but has been lacking due to experimental difficulties. We succeeded in the real-time mapping of the vibrational energy flow in a protein with the spatial resolution of a single amino acid residue by combining time-resolved resonance Raman spectroscopy and site-directed single-Trp mutagenesis. Anti-Stokes Raman intensities of the Trp residues at different sites exhibited different temporal evolutions, reflecting propagation of the energy released by the heme group. A classical heat transport model was not able to reproduce the entire experimental data set, showing that we need a molecular-level description to explain the energy flow in a protein. The systematic application of our general methodology to proteins with different structural motifs may provide a greatly increased understanding of the energy flow in proteins.

Protein conformational changes of the oxidative stress sensor, SoxR, upon redox changes of the [2Fe-2S] cluster probed with ultraviolet resonance Raman spectroscopy.

The [2Fe-2S] transcription factor, SoxR, a member of the MerR family, functions as a bacterial sensor of oxidative stress in Escherichia coli. SoxR is activated by reversible one-electron oxidation of the [2Fe-2S] cluster and enhances the production of various antioxidant proteins through the SoxRS regulon. Ultraviolet resonance Raman (UVRR) spectroscopic analysis of SoxR revealed conformational changes upon reduction of the [2Fe-2S] cluster in the absence and presence of promoter oligonucleotide. UVRR spectra reflected the environmental or structural changes of Trp following reduction. Notably, the environment around Trp91 contacting the [2Fe-2S] cluster was altered to become more hydrophilic, whereas that around Trp98 exhibited a small change to become more hydrophobic. In addition, changes in cation-π interactions between the [2Fe-2S] cluster and Trp91 were suggested. On the other hand, the environment around Tyr was barely affected by the [2Fe-2S] reduction. Binding of the promoter oligonucleotide triggered changes in Tyr located in the DNA-binding domain, but not Trp. Furthermore, conformational changes induced upon reduction of DNA-bound SoxR were not significantly different from those of DNA-free SoxR.

Intersubunit communication via changes in hemoglobin quaternary structures revealed by time-resolved resonance Raman spectroscopy: direct observation of the Perutz mechanism.

Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the β subunits. Changes in the resonance Raman spectra of the α heme and the β Ni-heme groups in the hybrid hemoglobin were observed upon CO photolysis in the α subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the ν(Fe-His) and the γ7 band of α heme was similar to that of unsubstituted hemoglobin, suggesting that substitution with Ni-heme did not perturb the allosteric dynamics of the hemoglobin. In the β subunits, no structural change in the Ni-heme was observed until 1 μs. In the microsecond regime, temporal evolution of the frequencies of the ν(Ni-His) and the γ7 band of β Ni-heme was observed concomitant with an R → T quaternary change at about 20 μs. The changes in the ν(Fe-His) and ν(Ni-His) frequencies of the α and β subunits with the common time constant of ∼20 μs indicated that the proximal tension imposed on the bond between the heme and the proximal histidine strengthened upon the quaternary changes in both the α and the β subunits concertedly. This observation is consistent with the Perutz mechanism for allosteric control of oxygen binding in hemoglobin and, thus, is the first real-time observation of the mechanism. Protein dynamics and allostery based on the observed time-resolved spectra also are discussed.

Direct Observation of the Structural Change of Tyr174 in the Primary Reaction of Sensory Rhodopsin II

Sensory rhodopsin II (SRII) is a negative phototaxis receptor containing retinal as its chromophore, which mediates the avoidance of blue light. The signal transduction is initiated by the photoisomerization of the retinal chromophore, resulting in conformational changes of the protein which are transmitted to a transducer protein. To gain insight into the SRII sensing mechanism, we employed time-resolved ultraviolet resonance Raman spectroscopy monitoring changes in the protein structure in the picosecond time range following photoisomerization. We used a 450 nm pump pulse to initiate the SRII photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, i.e., Trp76, Trp171, Tyr51, or Tyr174. The 225 nm UVRR spectra exhibited bleaching of the intensity for all the tryptophan bands within the instrumental response time, followed by a partial recovery with a time constant of 30 ps and no further changes up to 1 ns. In the 238 nm UVRR spectra, a fast recovering component was observed in addition to the 30 ps time constant component. A comparison between the spectra of the WT and Y174F mutant of SRII indicates that Tyr174 changes its structure and/or environment upon chromophore photoisomerization. These data represent the first real-time observation of the structural change of Tyr174, of which functional importance was pointed out previously.

Demonstration of a Light-Driven SO42– Transporter and Its Spectroscopic Characteristics

In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H+, Na+, and Cl-. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO42-, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl- or SO42-. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl- and SO42- with a subsequent secondary inward H+ movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl- and from 542 to 556 nm for SO42-, and the affinities of Cl- and SO42- were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO42-) transporter and report its spectroscopic characteristics.

Ultraviolet resonance Raman observations of the structural dynamics of rhizobial oxygen sensor FixL on ligand recognition.

FixL is a heme-based oxygen-sensing histidine kinase that induces expression of nitrogen fixation genes under hypoxic conditions. Oxygen binding to heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, inactivating autophosphorylation activity. Although FixL also can bind other diatomic ligands such as CO, the CO-bound FixL represents incomplete inhibition of kinase activity. Ultraviolet resonance Raman (UVRR) spectra revealed that oxygen binding to the truncated sensor domain of FixL markedly decreased the intensity of the Y8a band arising from Fα-10 Tyr. In contrast, no appreciable change in intensity of the Y8a band occurred after CO binding, and time-resolved UVRR spectra of the sensor domain of FixL upon O2 dissociation indicated that structural changes near Fα-10 Tyr occurred at ∼0.1 μs. These results suggest that O2 dissociation from FixL changes the protein conformation near the Fα-10 Tyr residue within a microsecond. The conformational changes of FixL upon O2 dissociation and the underlying sensing mechanism also are discussed.

Importance of Atomic Contacts in Vibrational Energy Flow in Proteins.

Vibrational energy flow in proteins was studied by monitoring the time-resolved anti-Stokes ultraviolet resonance Raman scattering of three myoglobin mutants in which a Trp residue substitutes a different amino acid residue near heme. The anti-Stokes Raman intensities of the Trp residue in the three mutants increased with similar rates after depositing excess vibrational energy at heme, despite the difference in distance between heme and each substituted Trp residue along the main chain of the protein. This indicates that vibrational energy is not transferred through the main chain of the protein but rather through atomic contacts between heme and the Trp residue. Distinct differences were observed in the amplitude of the band intensity change between the Trp residues at different positions, and the amplitude of the band intensity change exhibits a correlation with the extent of exposure of the Trp residue to solvent water. This correlation indicates that atomic contacts between an amino acid residue and solvent water play an important role in vibrational energy flow in a protein.