We have developed a spectrometer based on tunable quantum cascade lasers (QCLs) for recording time-resolved absorption spectra of proteins in the mid-infrared range. We illustrate its performance by recording time-resolved difference spectra of bacteriorhodopsin in the carboxylic range (1800-1700cm) and on the CO rebinding reaction of myoglobin (1960-1840cm), at a spectral resolution of 1cm. The spectrometric setup covers the time range from 4ns to nearly a second with a response time of 10-15ns. Absorption changes as low as 1×10 are detected in single-shot experiments at t>1μs, and of 5×10 in kinetics obtained after averaging 100 shots. While previous time-resolved IR experiments have mostly been conducted on hydrated films of proteins, we demonstrate here that the brilliance of tunable quantum cascade lasers is superior to perform ns time-resolved experiments even in aqueous solution (HO).

Immobilization of Cytochrome c oxidase (CcO) on electrodes makes voltage-driven reduction of oxygen to water possible. Efficient catalytic turnover in CcO/electrode systems is, however, often observed at large overpotentials that cannot be rationalized by the redox properties of the enzyme itself. To understand the structural basis for this observation, CcO was electrostatically adsorbed on amino-functionalized Ag electrodes, and the redox transitions of heme a and a3 were monitored via surface enhanced resonance Raman spectroscopy (SERRS) as a function of applied potential. Under completely anaerobic conditions, the reduction of heme a3 could be seen at potentials close to those measured in solution indicating an intact catalytic center. However, in the immobilized state, a new non-native heme species was observed that exhibited a redox potential much more negative than measured for the native hemes. Analysis of the high and low frequency SERR spectra indicated that this new species is formed from heme a upon axial loss of one histidine ligand. It is concluded that the formation of the non-native heme a species alters the potential-dependent electron supply to the catalytic reaction and, thus, can have a impact on the applicability of this enzyme in biofuel cells.

Cytochrome c oxidase (CcO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O2 reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CcO from Rhodobacter sphaeroides. The CO stretching frequency was monitored as a function of the electrical potential, using Fourier transform infrared (FTIR) absorption spectroelectrochemistry. We observed three different redox states (R4CO, R2CO, and O), determined their midpoint potential, and compared the resulting electric field to electrostatic calculations. A change in the local electric field strength of +2.9 MV/cm was derived, which was induced by the redox transition from R4CO to R2CO. We performed potential jump experiments to accumulate the R2CO and R4CO species and studied the FTIR difference spectra in the protein fingerprint region. The comparison of the experimental and computational results reveals that the key glutamic acid residue E286 is protonated in the observed states, and that its hydrogen-bonding environment is disturbed upon the redox transition of heme a3. Our experiments also suggest propionate A of heme a3 changing its protonation state in concert with the redox state of a second cofactor, heme a. This supports the role of propionic acid side chains as part of the proton-loading site.

Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe at the high temporal resolution inherent to IR spectroscopy.

We study the formation of a coordination network consisting of the organic pyridine-based 2,4,6-tris(4-pyridine)-1,3,5-triazine (T4PT) species and Cu atoms on Cu(111) and Ag(111) metal surfaces. Using scanning tunneling microscopy, we find that the organic molecule T4PT forms stable two-dimensional porous networks on the surface of Cu (111) and, by codeposition of Cu atoms, also on the Ag(111) crystal, in which Cu atoms are twofold coordinated by T4PT molecules. X-ray absorption spectroscopy measurements of the metal-organic network Cu-T4PT on Ag(111) accompanied by density-functional theory calculations show that the nitrogen atoms of the pyridine end groups of the T4PT molecules are the active sites in coordinating the Cu adatoms. X-ray magnetic circular dichroism experiments reveal that the Cu atom in such a metal-organic motif is in a low-valent d 10 state and has no magnetic moment.