In photosystem II (PSII), water is oxidized at the oxygen-evolving complex. This process occurs through a light-induced cycle that produces oxygen and protons. While coupled proton and electron ...transfer reactions play an important role in PSII and other proteins, direct detection of internal proton transfer reactions is challenging. Here, we demonstrate that the unnatural amino acid, 7-azatryptophan (7AW), has unique, pH-sensitive vibrational frequencies, which are sensitive markers of proton transfer. The intrinsically disordered, PSII subunit, PsbO, which contains a single W residue (Trp241), was engineered to contain 7AW at position 241. Fluorescence shows that 7AW-241 is buried in a hydrophobic environment. Reconstitution of 7AW(241)PsbO to PSII had no significant impact on oxygen evolution activity or flash-dependent protein dynamics. We conclude that directed substitution of 7AW into other structural domains is likely to provide a nonperturbative spectroscopic probe, which can be used to define internal proton pathways in PsbO.
Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally ...separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.
Metal substitution of heme proteins is widely applied in the study of biologically relevant electron transfer (ET) reactions. It has been shown that many modified proteins remain in their native ...conformation and can provide useful insights into the molecular mechanism of electron transfer between the native protein and its substrates. We investigated ET reactions between zinc-substituted cytochrome P450cam and small organic compounds such as quinones and ferrocene, which are capable of accessing the protein’s hydrophobic channel and binding close to the active site, like its native substrate, camphor. Following the substitution method developed by Gunsalus and co-workers Wagner, G. C., et al. (1981) J. Biol. Chem. 256, 6262–6265, we have identified two dominant forms of the zinc-substituted protein, F450 and F420, that exhibit different photophysical and photochemical properties. The ET behavior of F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and place themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that F420 corresponds to the open structure of the native cytochrome P450cam while F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450cam. The existence of two distinct conformers of Zn-bound P450cam is consistent with the findings of Goodin and co-workers Lee, Y.-T., et al. (2010) Biochemistry 49, 3412–3419 and has significant consequences for future electron transfer studies on this popular metalloenzyme.
Photoinduced electron transfer from S2 and S 1 states of azulene derivatives bound to TiO2 nanoparticles was investigated. Electron injection from S2 state occurred in < 100 fs and while that from S1 ...state occurred in < 50 fs. As in most TiO2-dye systems, charge recombination kinetics in all cases is heterogeneous implying that the process is dominated by trapping and de-trapping of excess electrons on TiO2 surfaces. Recombination resulting from electron injection from the S2 state of the directly bound dyes is up to ∼ 90% complete on a 120 ps timescale while that in tethered dyes is ∼50%. Back electron transfer resulting from S1 injection occurred on a much faster rate and is up to 70% complete on a 35 ps timescale. Surprisingly, no significant difference in the rates of recombination of species resulting from S1 injection was observed for directly bound and tethered dyes. Charge injection (140 ps) and recombination (45 ± 5% ns) observed between azulene incarcerated within a molecular container (hemicarcerand) and TiO2 are three decades slower than those observed in the directly bound and tethered dyes. The recombination kinetics observed in encapsulated azulene is homogenous strongly suggesting that tunneling of electrons from TiO2 through the wall of the hemicarcerand is the rate limiting step in the process. In the second part of the study, the potential use of fully encapsulating hemicarcerands in molecular transport was explored using zinc-substituted cytochrome c as a target molecule. The interaction between the encapsulated guests and the protein was probed by electron transfer quenching of the long-lived triplet phosphorescence of cytochrome c. The observed electron transfer rates (up to 1250 s-1) suggest that hemicarcerand binds in the vicinity of the exposed heme-edge of cytochrome c. In the last part of the project, preparation and characterization of the zinc analogue of cytochrome P450cam is presented. The protein is to be used in investigations on the use of hemicarcerand as a molecular shuttle to deliver electrons or holes from small hydrophobic guests across 22 Å substrate access channel of the target protein.
PDF
