Time-resolved Difference Spectra Research Articles

Time-resolved infrared difference spectroscopy in cells: Response of the basic region leucine zipper of aureochrome

Aureochromes are light, oxygen, voltage (LOV) proteins and central blue-light receptors in algae acting as light-gated transcription factors. The C-terminal LOV domain mediates blue-light recognition and the basic region leucine zipper (bZIP) domain binds a specific DNA motif as effector. LOV domains from aureochromes have been successfully applied in optogenetic tools. The light-induced response of aureochromes has been studied by a variety of biophysical techniques, but the mechanism of signal progression from LOV to bZIP remains unclear. We studied the bZIP-LOV module of aureochrome1a from the diatom Phaeodactylum tricornutum using time-resolved rapid-scan FTIR difference spectroscopy. Time-resolved difference spectra of bZIP-LOV in vitro revealed a time constant of 5 s for the formation of a light state dimer of the LOV domains and the concomitant loss of α-helical elements in the bZIP domain. To verify these observations in a near-native environment, in-cell infrared difference spectroscopy (ICIRD) was extended from a steady state to a time-resolved technique using LOV domains in bacterial cells. We established a time-resolved in-cell method with a resolution of 7.6 ms after the laser pulse. Using this technique, the response of bZIP-LOV was followed in living bacterial cells and the light-induced partial unfolding of bZIP was confirmed to take place in cells in a similar time range as in vitro. These results provide structural and kinetic insights into the signaling mechanism of aureochromes. The slow response points to an association of LOV to bZIP in the dark state prior to activation.

Identification of a Ubiquinone-Ubiquinol Quinhydrone Complex in Bacterial Photosynthetic Membranes and Isolated Reaction Centers by Time-Resolved Infrared Spectroscopy.

Ubiquinone redox chemistry is of fundamental importance in biochemistry, notably in bioenergetics. The bi-electronic reduction of ubiquinone to ubiquinol has been widely studied, including by Fourier transform infrared (FTIR) difference spectroscopy, in several systems. In this paper, we have recorded static and time-resolved FTIR difference spectra reflecting light-induced ubiquinone reduction to ubiquinol in bacterial photosynthetic membranes and in detergent-isolated photosynthetic bacterial reaction centers. We found compelling evidence that in both systems under strong light illumination-and also in detergent-isolated reaction centers after two saturating flashes-a ubiquinone-ubiquinol charge-transfer quinhydrone complex, characterized by a characteristic band at ~1565 cm-1, can be formed. Quantum chemistry calculations confirmed that such a band is due to formation of a quinhydrone complex. We propose that the formation of such a complex takes place when Q and QH2 are forced, by spatial constraints, to share a common limited space as, for instance, in detergent micelles, or when an incoming quinone from the pool meets, in the channel for quinone/quinol exchange at the QB site, a quinol coming out. This latter situation can take place both in isolated and membrane bound reaction centers Possible consequences of the formation of this charge-transfer complex under physiological conditions are discussed.

The Photoactive Excited State of the B12-Based Photoreceptor CarH.

We have used transient absorption spectroscopy in the UV-visible and X-ray regions to characterize the excited state of CarH, a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light. With visible excitation, a nanosecond-lifetime photoactive excited state is formed with unit quantum yield. The time-resolved X-ray absorption near edge structure difference spectrum of this state demonstrates that the excited state of AdoCbl in CarH undergoes only modest structural expansion around the central cobalt, a behavior similar to that observed for methylcobalamin rather than for AdoCbl free in solution. We propose a new mechanism for CarH photoreactivity involving formation of a triplet excited state. This allows the sensor to operate with high quantum efficiency and without formation of potentially dangerous side products. By stabilizing the excited electronic state, CarH controls reactivity of AdoCbl and enables slow reactions that yield nonreactive products and bypass bond homolysis and reactive radical species formation.

A quantum cascade laser setup for studying irreversible photoreactions in H2O with nanosecond resolution and microlitre consumption.

Time-resolved infrared spectroscopy on irreversible reactions requires in general an exchange of sample for thousands of acquisitions leading to high sample consumption. Here, we present a setup employing a modern quantum cascade laser (QCL) as a probe light source to record time-resolved difference spectra of irreversible photoreactions in H2O. The combination of the focused QCL with a pressure-tolerant flow cell and a micrometre stage orthogonal to the flow allowed us to drastically reduce the sample consumption. We investigated the irreversible photoreduction of the cofactor flavin mononucleotide (FMN) in H2O, which is a common reaction taking place in biological photoreceptors. A broad time range from 20 nanoseconds to 1 second was accessible, because the approach minimized any signal drift by the flow. Kinetics were recorded at 46 selected wavenumbers consuming 12 microlitres for a complete dataset. The tuning range of 1490-1740 cm-1 included relevant carbonyl vibrations and the region of strong water absorption at around 1650 cm-1. A continuous dataset in the spectral dimension was generated by applying a fit with a sum of Lorentzians. Subsequent global analysis allowed us to resolve reference spectra and kinetics of the photoreaction proceeding from the triplet excited state via the intermediate flavin anion radical to the product, the fully reduced state of FMN. Accordingly, the neutral radical state is not populated in the disproportionation. The approach strongly facilitates the spectroscopic access to irreversible reactions of flavin-containing photoreceptors and photoenzymes with high time resolution and small sample consumption.

Calculated vibrational properties of semiquinones in the A1 binding site in photosystem I

Time-resolved (P700+A1− – P700A1) FTIR difference spectra have been obtained using photosystem I (PSI) particles with several different quinones incorporated into the A1 protein binding site. Difference spectra were obtained for PSI with unlabeled and 18O labeled phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) and 2-methyl-1,4-naphthaquinone (2MNQ) incorporated, and for PSI with unlabeled 2,3-dimethyl-1,4-naphthoquinone (DMNQ) incorporated. (18O – 16O), (2MNQ – PhQ) and (DMNQ – PhQ) FTIR double difference spectra were constructed from the difference spectra. These double difference spectra allow one to more easily distinguish protein and pigment bands in convoluted difference spectra. To further aid in the interpretation of the difference spectra, particularly the spectra associated with the semiquinones, we have used two-layer ONIOM methods to calculate corresponding difference and double difference spectra. In all cases, the experimental and calculated double difference spectra are in excellent agreement. In previous two and three-layer ONIOM calculations it was not possible to adequately simulate multiple difference and double difference spectra. So, the computational approach outlined here is an improvement over previous calculations. It is shown that the calculated spectra can vary depending on the details of the molecular model that is used. Specifically, a molecular model that includes several water molecules that are near the incorporated semiquinones is required.

Photosystem I with benzoquinone analogues incorporated into the A1 binding site.

Time-resolved FTIR difference spectroscopy has been used to study photosystem I (PSI) particles with three different benzoquinones [plastoquinone-9 (PQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,3,5,6-tetrachloro-1,4-benzoquinone (Cl4BQ)] incorporated into the A1 binding site. If PSI samples are cooled in the dark to 77K, the incorporated benzoquinones are shown to be functional, allowing the production of time-resolved (P700+A1--P700A1) FTIR difference spectra. If samples are subjected to repetitive flash illumination at room temperature prior to cooling, however, the time-resolved FTIR difference spectra at 77K display contributions typical of the P700 triplet state (3P700), indicating a loss of functionality of the incorporated benzoquinones, that occurs because of double protonation of the incorporated benzoquinones. The benzoquinone protonation mechanism likely involves nearby water molecules but does not involve the terminal iron-sulfur clusters FA and FB. These results and conclusions resolve discrepancies between results from previous low-temperature FTIR and EPR studies on similar PSI samples with PQ incorporated.

Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10 ns to 1 s

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−1) and on the CO rebinding reaction of myoglobin (1960–1840cm−1), at a spectral resolution of 1cm−1. 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 1×10−4 are detected in single-shot experiments at t>1μs, and of 5×10−6 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 (H2O).

Does the singlet minus triplet spectrum with major photobleaching band near 680-682 nm represent an intact reaction center of Photosystem II?

We use both frequency- and time-domain low-temperature (5-20 K) spectroscopies to further elucidate the shape and spectral position of singlet minus triplet (triplet-bottleneck) spectra in the reaction centers (RCs) of Photosystem II (PSII) isolated from wild-type Chlamydomonas reinhardtii and spinach. It is shown that the shape of the nonresonant transient hole-burned spectrum in destabilized RCs from C. reinhardtii is very similar to that typically observed for spinach. This suggests that the previously observed difference in transient spectra between RCs from C. reinhardtii and spinach is not due to the sample origin but most likely due to a partial destabilization of the D1 and D2 polypeptides. This supports our previous assignments that destabilized RCs (referred to as RC680) (Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870), with a major photobleaching band near 680-682 nm and the absence of a photobleaching band near 673 nm, do not represent the intact RC residing within the PSII core complex. Time-resolved absorption difference spectra obtained for partially destabilized RCs of C. reinhardtii and for typical spinach RCs support the above conclusions. The absence of clear photobleaching bands near 673 and 684 nm (where the PD1 chlorophyll and the active pheophytin (PheoD1) contribute, respectively) in picosecond transient absorption spectra in both RCs studied in this work indicates that the cation can move from the primary electron donor (ChlD1) to PD1 (i.e., PD1ChlD1(+)PheoD1(-) → PD1(+)ChlD1PheoD1(-)). Therefore, we suggest that ChlD1 is the major electron donor in usually studied destabilized RCs (with a major photobleaching near 680-682 nm), although the PD1 path (where PD1 serves as the primary electron donor) is likely present in intact RCs, as discussed in Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870.

Kinetics and intermediates of the reaction of fully reduced Escherichia coli bo₃ ubiquinol oxidase with O₂.

Cytochrome bo₃ ubiquinol oxidase from Escherichia coli catalyzes the reduction of O₂ to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo₃ with O₂ with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 μs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 μs, 30 μs, 42 μs, 470 μs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 μs O₂ binding (3.8 × 10(7) M(-1) s(-1)), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o₃ F state is converted into the o₃(3+) oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O₂ binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O₂ reduction by bo₃, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Photochemistry of a dual-bacteriorhodopsin system in Haloarcula marismortui: HmbRI and HmbRII.

Recently, a dual-bacteriorhodopsin system, containing HmbRI and HmbRII, has been found in Haloarcula marismortui (Mol. Microbiol. 2013, 88, 551-561), and the light-driven proton pump activities were intrinsically different in a wide pH range. Compared with bacteriorhodopsin in H. salinarum (HsbR), the identical steady-state absorption contours of HsbR and HmbRs in the visible range indicated similarities in the retinal pocket. In addition, other reactive residues, including the proton relay channel, proton release group, and proton collecting funnel at the cytoplasm, were mostly conserved. We employed transient absorption spectroscopy and global analysis to characterize the photocycle intermediates and kinetics of HmbRI and HmbRII in the pH range of 4-8. The features of the time-resolved difference spectra of HmbRI indicated that the photocycle of HmbRI mainly followed the conventional pathway, including intermediates M, N, and O. A minute bypassed pathway from intermediate M needed to be included to better match the experimental data. The corresponding intermediate M' is attributed to the all-trans deprotonated Schiff base retinal, indicating the occurrence of retinal reisomerization prior to the reprotonation of the deprotonated Schiff base following the decay of intermediate M. Regarding HmbRII, its photocycle only followed the intermediates M and N, without intermediate O. The plausible molecular mechanisms, including the effects of the lengths of the loops and the distribution of the charged residues in the bacterio-opsin interior, were proposed to explain the differences in the photocycles. The pH-dependent photocycles were also investigated, and the results supported our proposed mechanism. Unravelling the photocycles of the HmbRs in the Haloarcula marismortui provided evidence that not only expanding the functional pH ranges but also the turnover kinetics are the strategies of the dual-bR system in the evolution of microbes in extreme environments.

Light-dark Adaptation of Channelrhodopsin C128T Mutant

Channelrhodopsins are microbial type rhodopsins that operate as light-gated ion channels. Largely prolonged lifetimes of the conducting state of channelrhodopsin-2 may be achieved by mutations of crucial single amino acids, i.e. cysteine 128. Such mutants are of great scientific interest in the field of neurophysiology because they allow neurons to be switched on and off on demand (step function rhodopsins). Due to their slow photocycle, structural alterations of these proteins can be studied by vibrational spectroscopy in more detail than possible with wild type. Here, we present spectroscopic evidence that the photocycle of the C128T mutant involves three different dark-adapted states that are populated according to the wavelength and duration of the preceding illumination. Our results suggest an important role of multiphoton reactions and the previously described side reaction for dark state regeneration. Structural changes that cause formation and depletion of the assumed ion conducting state P520 are only small and follow larger changes that occur early and late in the photocycle, respectively. They require only minor structural rearrangements of amino acids near the retinal binding pocket and are triggered by all-trans/13-cis retinal isomerization, although additional isomerizations are also involved in the photocycle. We will discuss an extended photocycle model of this mutant on the basis of spectroscopic and electrophysiological data.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-Type and EQ(I-286) Mutant of Rhodobacter sphaeroides Cytochrome c Oxidase

Cytochrome c oxidase from Rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. The O(2) reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound A), the P(R) state, the oxyferryl F form, and the oxidized state. In this study we investigated the reaction of dioxygen with the wild-type reduced R. sphaeroides cytochrome oxidase and the EQ(I-286) mutant using the CO flow-flash technique. Singular value decomposition and multiexponential fitting of the time-resolved optical absorption difference spectra showed that three apparent lifetimes, 18 μs, 53 μs, and 1.3 ms, are sufficient to fit the kinetics of the O(2) reaction of the wild-type enzyme. A comparison of the experimental intermediate spectra with the corresponding intermediate spectra of the bovine enzyme revealed that P(R) is not present in the reaction mechanism of the wild-type R. sphaeroides aa(3). Transient absorbance changes at 440 and 610 nm support this conclusion. For the EQ(I-286) mutant, in which a key glutamic residue in the D proton pathway is replaced by glutamine, two lifetimes, 16 and 108 μs, were observed. A spectral analysis of the intermediates shows that the O(2) reaction in the EQ(I-286) mutant terminates at the P(R) state, with 70% of heme a becoming oxidized. These results indicate significant differences in the kinetics of O(2) reduction between the bovine and wild-type R. sphaeroides aa(3) oxidases, which may arise from differences in the relative rates of internal electron and proton movements in the two enzymes.

Site-specific Protein Dynamics in Communication Pathway from Sensor to Signaling Domain of Oxygen Sensor Protein, HemAT-Bs: TIME-RESOLVED ULTRAVIOLET RESONANCE RAMAN STUDY

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ∼50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ∼50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.

Photoconversion Mechanism of a Green/Red Photosensory Cyanobacteriochrome AnPixJ: Time-Resolved Optical Spectroscopy and FTIR Analysis of the AnPixJ-GAF2 Domain

The photoconversion mechanism of a green/red sensory cyanobacteriochrome AnPixJ was studied. The phycocyanobilin-binding second GAF domain of AnPixJ of Anabaena sp. PCC 7120 was expressed in Escherichia coli cells. The His-tagged AnPixJ-GAF2 domain exhibited photoconversion between the green- and red-absorbing forms, APg(543) and APr(648), respectively. We detected four intermediate states in the photocycle between them, as follows: APr(648) → red light → APr(648)* → (with a rise time constant τ(r) of <100 ns) R1(650-80) (with a decay time constant τ(d) of <1 μs) → R2(610) (τ(d) = 920 μs) → APg(543) → green light → APg(543)* → (τ(r) < 50 ns) G1(570) (τ(d) = 190 μs) → G2(630) (τ(d) = 1.01 ms) → APr(648). These intermediates were named for their absorption peak wavelengths, which were estimated on the basis of the time-resolved difference spectra and global analysis of the time courses. The absorption spectrum of APr(648) resembles that of the Pr form of the phytochrome, while all the other states showed peaks at 530-650 nm and had wider bandwidths with smaller peak amplitudes. The fastest decay phases of fluorescence from APr(648)* and APg(543)* gave lifetimes of 200 and 42 ps, respectively, suggesting fast primary reactions. The APg(543)-minus-APr(648) difference FTIR spectrum in an H(2)O medium was significantly different from those reported for the Pfr/Pr difference spectra in phytochromes. Most of the peaks in the difference spectrum were shifted in the D(2)O medium, suggesting the high accessibility to the aqueous phase. The interactions of the phycocyanobilin chromophore with the surrounding amino acid residues, which are fairly different from those in the GAF domain of phytochromes, realize the unique green/red photocycle of AnPixJ.

Rhodopsin in Nanodiscs Has Native Membrane-like Photointermediates

Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I(480) ⇄ Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.

On the mechanism of formation and spectral properties of radical anions generated by the reduction of –[Re I(CO) 3(5-nitro-1,10-phenanthroline)] + and –[Re I(CO) 3(3,4,7,8-tetramethyl-1,10-phenanthroline)] + pendants in poly-4-vinylpyridine polymers

The electrochemical reduction in aprotic media of –[Re I(CO) 3L] + pendants in poly-4-vinylpyridine polymers is compared to that of [Re I(CO) 3L] + complexes (L = 5-nitro-1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline). The UV–Vis absorption spectra of the reduced radical anions of 5-nitro-1,10-phenanthroline (NO 2-phen) and 3,4,7,8-tetramethyl-1,10-phenanthroline (tmphen) were obtained by spectro-electrochemistry of [Re I(CO) 3(NO 2-phen)(CH 3CN)] + and [Re I(CO) 3(tmphen)(CH 3CN)] +, respectively. Similar spectra were obtained for the radical anions NO 2 - -phen and tmphen − after pulse radiolysis experiments with –[Re I(CO) 3L] +-containing polymers. The analysis of the time-resolved difference spectra was performed using “multivariate curve resolution” (MCR) techniques. Unlike e solv - , C H 2OH radicals were unable to reduce tmphen ligands. The reaction of e solv - and/or C H 2OH with –[Re I(CO) 3(NO 2-phen)] +-containing polymers generates –[Re I(CO) 3( NO 2 - -phen)] pendants which after disproportionation give rise to products with λ max = 380 nm. The kinetic behavior of –[Re I(CO) 3( NO 2 - -phen)] pendants under different experimental conditions is discussed.

Effect of Molecular Assembly on Photocycle of Reconstituted Bacteriorhodopsin: Significant Blue Shift of the Late M Photointermediate in the Liquid Crystalline Phase

Abstract The photocycle of bacteriorhodopsin (bR) reconstituted into dimyristoylphosphatidylcholine vesicles was investigated with transient visible absorption spectroscopy. The measured time-resolved difference spectra indicated that two substates of the M intermediate with almost the same absorption maximum were observed in the gel state, whereas the spectrum of M showed a splitting into an early and a late component shifted by approximately 15 nm in the liquid crystalline phase, suggesting disassembly of bR molecules induces remarkable structural changes around the retinal pocket of the late M intermediate responsible for switching protein conformation from a proton release form to a proton uptake form.

Direct Deconvolution of Two-State Pump-Probe X-ray Absorption Spectra and the Structural Changes in a 100 ps Transient of Ni(II)-tetramesitylporphyrin

Full multiple scattering (FMS) Minuit XANES (MXAN) has been combined with laser pump-probe K-edge X-ray absorption spectroscopy (XAS) to determine the structure of photoexcited Ni(II)tetramesitylporphyrin, Ni(II)TMP, in dilute toluene solution. It is shown that an excellent simulation of the XANES spectrum is obtained, excluding the lowest-energy bound-state transitions. In ground-state Ni(II)TMP, the first-shell and second-shell distances are, respectively, d(Ni-N) = (1.93 +/- 0.02) A and d(Ni-C) = (2.94 +/- 0.03) A, in agreement with a previous EXAFS result. The time-resolved XANES difference spectrum was obtained (1) from the spectra of Ni(II)TMP in its photoexcited T(1) state and its ground state, S(0). The XANES difference spectrum has been analyzed to obtain both the structure and the fraction of the T(1) state. If the T(1) fraction is kept fixed at the value (0.37 +/- 0.10) determined by optical transient spectroscopy, a 0.07 A elongation of the Ni-N and Ni-C distances [d(Ni-N) and d(Ni-C)] is found, in agreement with the EXAFS result. However, an evaluation of both the distance elongation and the T(1) fraction can also be obtained using XANES data only. According to experimental evidence, and MXAN simulations, the T(1) fraction is (0.60 +/- 0.15) with d(Ni-N) = (1.98 +/- 0.03) A (0.05 A elongation). The overall uncertainty of these results depends on the statistical correlation between the distances and T(1) fraction, and the chemical shift of the ionization energy because of subtle changes of metal charge between the T(1) and S(0) states. The T(1) excited-state structure results, independently obtained without the excited-state fraction from optical transient spectroscopy, are still in agreement with previous EXAFS investigations. Thus, full multiple scattering theory applied through the MXAN formalism can be used to provide structural information, not only on the ground-state molecules but also on very short-lived excited states through differential analysis applied to transient photoexcited species from time-resolved experiments.

The pH Dependence of Heme Pocket Hydration and Ligand Rebinding Kinetics in Photodissociated Carbonmonoxymyoglobin

We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation ( Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254-1259 ). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change.

Active Internal Waters in the Bacteriorhodopsin Photocycle. A Comparative Study of the L and M Intermediates at Room and Cryogenic Temperatures by Infrared Spectroscopy

We present time-resolved room-temperature infrared difference spectra for the bacteriorhodopsin (bR) photocycle at 8 cm (-1) spectral and 5 micros temporal resolution, from 4000 to 800 cm (-1). An in situ hydration method allowed for a controlled and stable sample hydration (92% relative humidity), largely improving the quality of the data without affecting the functionality of bR. Experiments in both H 2 (16)O and H 2 (18)O were conducted to assign bands to internal water molecules. Room-temperature difference spectra of the L and M intermediates minus the bR ground state (L-BR and M-BR, respectively) were comprehensively compared with their low-temperature counterparts. The room-temperature M-BR spectrum was almost identical to that obtained at 230 K, except for a continuum band. The continuum band contains water vibrations from this spectral comparison between H 2 (16)O and H 2 (18)O, and no continuum band at 230 K suggests that the protein/solvent dynamics are insufficient for deprotonation of the water cluster. On the other hand, an intense positive broadband in the low-temperature L-BR spectrum (170 K) assigned to the formation of a water cavity in the cytoplasmic domain is absent at room temperature. This water cavity, proposed to be an essential feature for the formation of L, seems now to be a low-temperature artifact caused by restricted protein dynamics at 170 K. The observed differences between low- and room-temperature FTIR spectra are further discussed in light of previously reported dynamic transitions in bR. Finally, we show that the kinetics of the transient heat relaxation of bR after photoexcitation proceeds as a thermal diffusion process, uncorrelated with the photocycle itself.