NADH model systems functionalized with Zn(II)-cyclen as flavin binding site-structure dependence of the redox reaction within reversible aggregates

The relative positions and conformations of the prosthetic group FAD and the cofactor NADH have been remarkably conserved within the structurally diverse group of flavin enzymes. To provide a chemical rational for such an obviously optimal relative disposition of the redox partners for efficient reaction we have synthesized NADH models with Zn(II)-cyclen substituents for reversible flavin binding in water. Altogether, four of these model systems with systematically varying spacer length between the recognition site and the redox active dihydronicotinamide were prepared. The binding of these model systems to riboflavin tetraacetate was confirmed by potentiometric pH titration in water and their reaction with flavin was followed by UV-vis spectroscopy in aqueous media under physiological conditions. The measurements reveal a significant rate enhancement of up to 175 times that of an intermolecular reaction. Moreover, a strong dependence of the reaction rate on the spacer length was observed, which clearly shows that within the dynamic reversible assembly only the optimal relative disposition of the redox partners ensures an efficient redox reaction.     

Unraveling the flavin-catalyzed photooxidation of benzylic alcohol with transient absorption spectroscopy from sub-pico- to microseconds

Flavin-mediated photooxidations have been described for applications in synthetic organic chemistry for some time and are claimed to be a route to the use of solar energy. We present a detailed investigation of the involved photophysical and photochemical steps in methoxybenzyl alcohol oxidation on a timescale ranging from sub-picoseconds to tens of microseconds. The results establish the flavin triplet state as the key intermediate for the photooxidation. The initial step is an electron transfer from the alcohol to the triplet state of the flavin catalyst with (3)k(ET)≈ 2 × 10(7) M(-1) s(-1), followed by a proton transfer in ～6 μs. In contrast, the electron transfer involving the singlet state of flavin is a loss channel. It is followed by rapid charge recombination (τ = 50 ps) without significant product formation as seen when flavin is dissolved in pure benzylic alcohol. In dilute acetonitrile/water solutions of flavin and alcohol the electron transfer is mostly controlled by diffusion, though at high substrate concentrations >100 mM we also find a considerable contribution from preassociated flavin-alcohol-aggregates. The model including a productive triplet channel and a competing singlet loss channel is confirmed by the course of the photooxidation quantum yield as a function of substrate concentration: We find a maximum quantum yield of 3% at 25 mM of benzylic alcohol and significantly smaller values for both higher and lower alcohol concentrations. The observations indicate the importance to perform flavin photooxidations at optimized substrate concentrations to achieve high quantum efficiencies and provide directions for the design of flavin photocatalysts with improved performance.     

A conclusive mechanism of the photoinduced reaction cascade in blue light using flavin photoreceptors

On the basis of extensive first-principle calculations within the framework of quantum mechanics/molecular mechanics (QM/MM), a conclusive mechanism for the formation of the signaling state of blue light using flavin (BLUF) domain proteins is proposed which is compatible with the experimental data presently available. Time-dependent density functional, as well as advanced coupled cluster response theory was employed for the QM part in order to describe the relevant excited states. One of the key residues involved in the mechanism is the glutamine adjacent to the flavin chromophore. The reaction cascade, triggered by the initial photoexcitation of the flavin chromophore, involves isomerization of this residue but no rotation as assumed previously. The fact that only the environment, but not the flavin chromophore by itself, is chemically transformed along the individual steps of the mechanism is unique for biological photoreceptors. The final isomer of the glutamine tautomer, i.e., the imidic acid, is further stabilized by the interchange of a methionine residue in the binding pocket with a tryptophan residue. The flip of these two residues might be the trigger for the large conformational change of this protein which is consequently transmitted as the signal to the biological environment.     

De novo design, synthesis, and function of semiartificial myoglobin conjugated with coiled-coil two-alpha-helix peptides

The introduction of a flavin chromophore on the myoglobin (Mb) surface and an effective electron-transfer (ET) reaction through the flavin were successfully achieved by utilizing the self-assembly of heterostranded coiled-coil peptides. We have prepared a semiartificial Mb, named Mb-1alphaK, in which an amphiphilic and cationic alpha-helix peptide is conjugated at the heme propionate (Heme-1alphaK). Heme-1alphaK has a covalently bound iron-protoporphyrin IX (heme) at the N terminus of a 1alphaK peptide sequence. This sequence was designed to form a heterostranded coiled-coil in the presence of a counterpart amphiphilic and anionic 1alphaE peptide sequence in a parallel orientation. Two peptides, Fla(1)-1alphaE and Fla(31)-1alphaE, both incorporating a 10-methylisoalloxazine moiety as an artificial flavin molecule, were also prepared (Fla=2-[7-(10-methyl)isoalloxazinyl]-2-oxoethyl). Heme-1alphaK was successfully inserted into apomyoglobin to give Mb-1alphaK. Mb-1alphaK recognized the flavin-modified peptides and a two-alpha-helix structure was formed. In addition, an efficient ET from reduced nicotinamide adenine dinucleotide to the heme center through the flavin unit was observed. The ET rate was faster in the presence of Fla(1)-1alphaE than in the presence of Fla(31)-1alphaE or the equivalent molecule that has no peptide chain. These results demonstrate that the introduction of a functional chromophore on the Mb surface can be achieved by using specific peptide-peptide interactions. Moreover, the dependence of the ET rate on the position of the flavin indicated that the distance between the heme active site and the flavin chromophore was regulated by the three-dimensional structure of the designed polypeptide.     

A FLAVOPROTEIN MAY MEDIATE THE BLUE LIGHT-ACTIVATED BINDING OF GUANOSINE 5'-TRIPHOSPHATE TO ISOLATED PLASMA MEMBRANES OF Pisum sativum L.*

Abstract— A blue light photoreceptor has not been identified in higher plants. Most proposals for a blue light-absorbing chromophore lack evidence for a direct connection between the putative chromophdre and a biological effect. Fluorescence data for the plasma membrane from etiolated buds of Pisum sativum L. suggest that we are measuring fluorescence emission of flavin species, and probably not pterin species. Fluorescence data indicate that a putative flavin exists associated with a protein or protein complex in the plasma membrane. Excitation of plasma membranes that were boiled in the presence of 0.1% sodium dodecyl sulfate and treated with blue light yields a fluorescence band with a maximum of approximately 552 nm. This fluorescence emission can be rapidly quenched by the flavin antagonists phenylacetic acid (PAA) and KI. Blue light-enhanced binding of guanosine 5'-[Γ-thio]triphosphate (GTPγS) to a protein in the plasma membrane is strongly inhibited by PAA, KI, and NaN₃, all quenchers of flavin excited states, indicating that a chromophore for this photoreaction may be a flavin associated with a plasma membrane protein. The above evidence is consistent with the participation of a flavin as the chromophore for the light-induced GTP-binding reaction in pea plasma membrane.     

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.     

Synchronous photoinitiation of endothelial NO synthase activity by a nanotrigger targeted at its NADPH site

We designed a new nanotrigger to synchronize and monitor an enzymatic activity interacting specifically with the conserved NADPH binding site. The nanotrigger (NT) combines a docking moiety targeting the NADPH site and a chromophore moiety responsive to light excitation for efficient electron transfer to the protein. Specific binding of the nanotrigger to the reductase domain of the endothelial nitric oxide synthase (eNOSred) was demonstrated by competition between NADPH and the nanotrigger on the reduction of eNOSred flavin. A micromolar Ki was estimated. We had monitored initiation of eNOSred activity by ultrafast transient spectroscopy. The transient absorption spectrum recorded at 250 ps fits the expected sum of the reduced and oxidized species, independently obtained by other chemical methods, in agreement with a photoinduced electron transfer from the excited nanotrigger to the flavin moiety of eNOSred. The rate of electron transfer from the excited state of the nanotrigger (NT*) to the protein is estimated to be k(ET) = (7 +/- 2) x 10(9) s(-1) using the decay of oxidized eNOSred-bound nanotrigger compared against prereduced eNOSred or glucose 6-P dehydrogenase as controls. This fast electron transfer bypasses the slow hydride transfer to initiate NOS catalysis as shown by ultrafast kinetics using the eNOSred mutated in the regulatory F1160 residue. The selective targeting of the nanotrigger to NADPH sites should allow controlled initiation of the enzymatic activity of numerous proteins containing an NADPH site.     

Structural chemistry of a green fluorescent protein Zn biosensor

We designed a green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) (enhancing fluorescence intensity) and Cu(II) (quenching fluorescence) directly to a chromophore ligand that resembles a dipyrrole unit of a porphyrin. Crystallographic structure determination of apo, Zn(II)-bound, and Cu(II)-bound BFPms1 to better than 1.5 A resolution allowed us to refine metal centers without geometric restraints, to calculate experimental standard uncertainty errors for bond lengths and angles, and to model thermal displacement parameters anisotropically. The BFPms1 Zn(II) site (KD = 50 muM) displays distorted trigonal bipyrimidal geometry, with Zn(II) binding to Glu222, to a water molecule, and tridentate to the chromophore ligand. In contrast, the BFPms1 Cu(II) site (KD = 24 muM) exhibits square planar geometry similar to metalated porphyrins, with Cu(II) binding to the chromophore chelate and Glu222. The apo structure reveals a large electropositive region near the designed metal insertion channel, suggesting a basis for the measured metal cation binding kinetics. The preorganized tridentate ligand is accommodated in both coordination geometries by a 0.4 A difference between the Zn and Cu positions and by distinct rearrangements of Glu222. The highly accurate metal ligand bond lengths reveal different protonation states for the same oxygen bound to Zn vs Cu, with implications for the observed metal ion specificity. Crystallographic anisotropic thermal factor analysis validates metal ion rigidification of the chromophore in enhancement of fluorescence intensity upon Zn(II) binding. Thus, our high-resolution structures reveal how structure-based design has effectively linked selective metal binding to changes in fluorescent properties. Furthermore, this protein Zn(II) biosensor provides a prototype suitable for further optimization by directed evolution to generate metalloprotein variants with desirable physical or biochemical properties.     

Theoretical study on the repair mechanism of the (6-4) photolesion by the (6-4) photolyase

UV irradiation of DNA can lead to the formation of mutagenic (6-4) pyrimidine-pyrimidone photolesions. The (6-4) photolyases are the enzymes responsible for the photoinduced repair of such lesions. On the basis of the recently published crystal structure of the (6-4) photolyase bound to DNA [Maul et al. 2008] and employing quantum mechanics/molecular mechanics techniques, a repair mechanism is proposed, which involves two photoexcitations. The flavin chromophore, initially being in its reduced anionic form, is photoexcited and donates an electron to the (6-4) form of the photolesion. The photolesion is then protonated by the neighboring histidine residue and forms a radical intermediate. The latter undergoes a series of energy stabilizing hydrogen-bonding rearrangements before the electron back transfer to the flavin semiquinone. The resulting structure corresponds to the oxetane intermediate, long thought to be formed upon DNA-enzyme binding. A second photoexcitation of the flavin promotes another electron transfer to the oxetane. Proton donation from the same histidine residue allows for the splitting of the four-membered ring, hence opening an efficient pathway to the final repaired form. The repair of the lesion by a single photoexcitation was shown not to be feasible.     

Absorption and fluorescence spectroscopic characterisation of a phenothiazine–flavin dyad

The phenothiazine–phenylene–isoalloxazine dyad, 3-methyl-10-[4-(10-heptyl-10H-phenothiazin-3-yl)-phenyl]-10H-benzo[g]pteridine-2,4-dione, dissolved in either dichloromethane or acetonitrile is characterized by absorption and emission spectroscopy. Absorption cross-section spectra, stimulated emission cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and degrees of fluorescence polarisation are determined. The fluorescence decay is determined by time-resolved measurements. The dye photo-stability is investigated by observation of absorption spectral changes due to prolonged blue-light excitation. The absorption spectrum of the dyad resembles the superposition of the absorption of the isoalloxazine (flavin) moiety and of the phenylphenothiazine moiety. Photo-excitation of the flavin moiety causes fluorescence quenching by ground-state reductive electron transfer from phenylphenothiazine to isoalloxazine followed by charge recombination. Photo-excitation of the phenothiazine moiety causes (i) efficient excited-state oxidative electron transfer from phenothiazine to isoalloxazine with successive recombination, and (ii) moderate energy transfer followed by ground-state phenothiazine electron transfer and recombination.     

Photoisomerization of the green fluorescence protein chromophore and the meta- and para-amino analogues

The Z --> E photoisomerization and fluorescence quantum yields for the wild-type green fluorescence protein (GFP) chromophore (p-HBDI) and its meta- and para-amino analogues (m-ABDI and p-ABDI) in aprotic solvents (hexane, THF, and acetonitrile) and protic solvents (methanol and 10-20% H(2)O in THF) are reported. The dramatic decrease in the quantum yields on going from aprotic to protic solvents indicates the important role of solvent-solute hydrogen bonding in the nonradiative decay pathways. The enhanced fluorescence of m-ABDI is also discussed.     

What is Best Strategy for Water Soluble Fluorescence Dyes?—A Case Study Using Long Fluorescence Lifetime DAOTA Dyes**

The lipophilic nature of organic dyes complicates their effectiveness in aqueous solutions. In this work we investigate three different strategies for achieving water-solubility of the diazaoxatriangulenium (DAOTA⁺) chromophore: hydrophilic counter ions, aromatic sulfonation of the chromophore, and attachment of charged side chains. The long fluorescence lifetime (FLT, τ_f=20 ns) of DAOTA⁺ makes it a sensitive probe to analyze solvation and aggregation effects. Direct sulfonation of the chromophore was found to increase solubility drastically, but at the cost of greatly reduced quantum yields (QYs) due to enhanced non-radiative deactivation processes. The introduction of either cationic (4) or zwitterionic side chains (5), however, brings the FLT (τ_f=18 ns) and QY (ϕ_f=0.56) of the dye to the same level as the parent chromophore in acetonitrile. Time-resolved fluorescence spectroscopy also reveals a high resistance to aggregation and non-specific binding in a high loading of bovine serum albumin (BSA). The results clearly show that addition of charged flexible side chains is preferable to direct sulfonation of the chromophore core.     

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.     

Thiourea-enhanced flavin photooxidation of benzyl alcohol

Upon irradiation, flavin oxidises 4-methoxybenzyl alcohol to the corresponding aldehyde using aerial O(2) as the terminal oxidant. We have observed that this reaction is significantly accelerated by the presence of thiourea. A series of thiourea-functionalised flavins has been prepared from flavin isothiocyanates and their photocatalytic efficiencies have been monitored by NMR. The alcohol photooxidation proceeds rapidly and cleanly with high turnover numbers of up to 580, exceeding previously reported performances. A likely mechanistic rationale for the more than 30-fold acceleration of the photo-redox reaction by thiourea has been derived from spectroscopic, electrochemical, and kinetic studies. Thus, thiourea acts as an electron-transfer mediator for the initial photooxidation of 4-methoxybenzyl alcohol by the excited flavins. This mechanism has similarities to electron-relay mechanisms in flavoenzymes, for which cysteine sulfenic acid intermediates are proposed. The observation that thiourea mediates flavin photo-redox processes is valuable for the design of more sophisticated photocatalysts based on Nature's best redox chromophore.     

BLUE AND ULTRAVIOLET-B LIGHT PHOTORECEPTORS IN PARSLEY CELLS

Abstract— Ultraviolet-B (UV-B) and blue light photoreceptors have been shown to regulate chalcone synthase and flavonoid synthesis in parsley cell cultures. These photoreceptors have not yet been identified. In the present work, we studied UV-B photoreception with physiological experiments involving temperature shifts and examined the possible role of flavin in blue and UV-B light photoreception. Cells irradiated with UV-B light (0.5–15 min) at 2°C have the same fluence requirement for chalcone synthase and flavonoid induction as controls irradiated at 25°C. This is indicative of a purely photochemical reaction. Cells fed with riboflavin and irradiated with 6 h of UV-containing white light synthesize higher levels of chalcone synthase and flavonoid than unfed controls. This effect did not occur with blue light. These results indicate that flavin-sensitization requires excitation of flavin and the UV-B light photoreceptor. The in vivo kinetics of flavin uptake and bleaching indicate that the added flavin may act at the surface of the plasma membrane. In view of the likely role of membrane-associated flavin in photoreception, we measured in vitro flavin binding to microsomal membranes. At least one microsomal flavin binding site was solubilized by resuspension of a microsomal pellet in buffer with high KPi and NaCl concentrations and centrifugation at 38000 g. The 38000 g insoluble fraction had much greater flavin binding and contained a receptor with an apparent K_D of about 3.6 μM and an estimated in vivo concentration of at least 6.7 × 10–⁸M. Flavin mononucleotide, roseoflavin, and flavin adenine dinucleotide can compete with riboflavin for this binding site(s), although each has lower affinity than riboflavin. Most microsomal protein was solubilized by resuspension of the microsomal pellet in non-denaturing detergents and centrifugation at 38 000 g ; however, this inhibited flavin binding, presumably because of disruption of the environment of the flavin receptor. The parsley microsomal flavin binding receptor(s) have a possible role in physiological photoreception.     

Photoexcitation of the blue light using FAD photoreceptor AppA results in ultrafast changes to the protein matrix

Photoexcitation of the flavin chromophore in the BLUF photosensor AppA results in a conformational change that leads to photosensor activation. This conformational change is mediated by a hydrogen-bonding network that surrounds the flavin, and photoexcitation is known to result in changes in the network that include a strengthening of hydrogen bonding to the flavin C4═O carbonyl group. Q63 is a key residue in the hydrogen-bonding network, and replacement of this residue with a glutamate results in a photoinactive mutant. While the ultrafast time-resolved infrared (TRIR) spectrum of Q63E AppA(BLUF) is characterized by flavin carbonyl modes at 1680 and 1650 cm(-1), which are similar in frequency to the analogous modes from the light activated state of the wild-type protein, a band is also observed in the TRIR spectrum at 1724 cm(-1) that is unambiguously assigned to the Q63E carboxylic acid based on U-(13)C labeling of the protein. Light absorption instantaneously (<100 fs) bleaches the 1724 cm(-1) band leading to a transient absorption at 1707 cm(-1). Because Q63E is not part of the isoalloxazine electronic transition, the shift in frequency must arise from a sub picosecond perturbation to the flavin binding pocket. The light-induced change in the frequency of the Q63E side chain is assigned to an increase in hydrogen-bond strength of 3 kcal mol(-1) caused by electronic reorganization of the isoalloxazine ring in the excited state, providing direct evidence that the protein matrix of AppA responds instantaneously to changes in the electronic structure of the chromophore and supporting a model for photoactivation of the wild-type protein that involves initial tautomerization of the Q63 side chain.     

Enantiospecific assay for mammalian carbonyl reductase by liquid chromatography with fluorescence detection

A fluorogenic substrate with an unsymmetrical carbonyl for the sensitive assay of mammalian carbonyl reductase activities, 4-(6-methoxy-2-benzoxazolyl)acetophenone (I), has been prepared. The fluorescence quantum yield of I in acetonitrile is 0.12 at the emission maximum of 448 nm. The corresponding racemic alcohol produced by the chemical reduction of I, (+/-)-sec.-[4-(6-methoxy-2-benzoxazolyl)]phenethyl alcohol (II), exhibits ca. three- to fourteen-fold higher fluorescence at a shorter wavelength emission maximum of 370 nm in conventional solvents. Each enantiomer of II is sufficiently resolved on a chiral cellulose high-performance liquid chromatographic column without derivatization and quantified with high reproducibility. The detection limit for II is 20 fmol per injection at a signal-to-noise ratio of 3. The validity and applicability of I are evaluated with cytosols of mammalian tissues. The optimal pH for metabolic reduction of I in rabbit liver cytosol preparations is 6.2 in the presence of NADPH. The metabolism is proved to be highly stereoselective. The resulting alcohol produced by mammalian tissue preparations, except rabbit kidney, is predominantly of the S-(-)-configuration.     

Why BLUF photoreceptors with roseoflavin cofactors lose their biological functionality

The photophysics of roseoflavin in three different environments is investigated by using ab initio and quantum mechanics/molecular mechanics methods. Intramolecular charge transfer is shown to be responsible for the quenching of the fluorescence in the gas phase, and in the water environment. However, for the roseoflavin incorporated into the blue light using flavin (BLUF) protein environment (substituting the native flavin) no such deactivation is found. The conical intersection between the locally excited state of the chromophore and the charge transfer state involving the tyrosine residue, which in the native BLUF domain is responsible for initiating the photocycle, is missing for the roseoflavin substituted protein. This explains the experimental observations of the lack of any photocycle, and the loss of the biological function of the BLUF photoreceptor reported earlier.     

Synthesis of pyrene—acridine bis-intercalators and effects of binding to DNA

A bis-intercalating compound containing pyrene and 9-aminoacridine chromophores (N-(5-(1-pyrenyl)-pentyl)-6-(9-acridinylamino) hexylamide, I), was prepared and its interaction with double-stranded DNA was investigated. Homologous compounds in which the two chromophores were connected by a linear carbon chain (pentamethylene (II), tetramethylene (III) and methylene (IV)) were also prepared. In acetonitrile solutions of the free ligands, the presence of the proximal pyrene results in reduced acridine fluorescence relative to 9methylaminoacridine (9-MAA), and the degree of quenching increases with decreasing chain length. The quenching process is assigned to exothermic electron transfer from pyrene to the excited 9-aminoacridine (9-AA) chromophore. In the presence of DNA, the relative quenching order is reversed, and I and IV are quenched more strongly than II and III. From linear dichroism experiments, it is concluded that I binds by bis-intercalation of the pyrene and acridine moieties, III and IV undergo intercalation of the acridine chromophore and II binds by partial bis-intercalation at two contiguous sites.     

Role of hydrogen-bond network in energy storage of bacteriorhodopsin's light-driven proton pump revealed by ab initio normal-mode analysis

Vibrational modes of the hydrogen-bond network in the binding site of bacteriorhodopsin (bR), a protein in halobacteria functioning as a light-driven proton pump, were investigated by an ab initio quantum mechanical/molecular mechanical (QM/MM) method. Normal-mode analysis calculations for O-D and N-D stretching modes of internal water molecules and the Schiff base of the retinal chromophore in the early intermediate state, K, reproduced well experimentally observed vibrational spectra. Supported by agreement with observed spectra, the QM/MM calculation suggests that weakened hydrogen bonds upon photoisomerization of the chromophore are an important means of energy storage in bR.