Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family.

Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH· state upon binding to a CPD, and, in the absence of substrate, the rate of FADH· oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802-2805, 2015). Here, we present the pH-dependence of the oxidation of FADH· by ferricyanide, which revealed a switch between slow and fast oxidation with a pKa ≈ 7.0. Stopped-flow mixing was used to measure the oxidation of FADH- to FADH· at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped flow, but there was only a small effect of pH. In addition, resonance Raman measurements of FADH· in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH· oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-effect on the FADH· oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH· and give rise to the pH-effect.

Enzyme-Substrate Binding Kinetics Indicate That Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer.

Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) that are formed on DNA upon exposure of cells to ultraviolet light. The light-driven electron-transfer mechanism by which photolyase catalyzes the CPD monomerization after the enzyme-substrate complex has formed has been studied extensively. However, much less is understood about how photolyase recognizes CPDs on DNA. It has been clearly established that photolyase, like many other DNA-repair proteins, requires flipping of the CPD site into an extrahelical position. Photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base. In this paper, we perform direct measurements of photolyase binding to CPD-containing undecamer DNA that has been labeled with a fluorophore. We find that the association constant of ∼2 × 10(6) M(-1) is independent of the location of the CPD on the undecamer DNA. The binding kinetics of photolyase are best described by two rate constants. The slower rate constant is ∼10(4) M(-1) s(-1) and is most likely due to steric interference of the fluorophore during the binding process. The faster rate constant is on the order of 2.5 × 10(5) M(-1) s(-1) and reflects the binding of photolyase to the CPD on the DNA. This result indicates that photolyase finds and binds to a CPD lesion 100-4000 times slower than other DNA-repair proteins. In light of the existing literature, we propose a mechanism in which photolyase recognizes a CPD that is flipped into an extrahelical position via a three-dimensional search.

Evidence for concerted electron proton transfer in charge recombination between FADH- and 306Trp• in Escherichia coli photolyase.

Proton-coupled electron-transfer (PCET) is a mechanism of great importance in protein electron transfer and enzyme catalysis, and the involvement of aromatic amino acids in this process is of much interest. The DNA repair enzyme photolyase provides a natural system that allows for the study of PCET using a neutral radical tryptophan (Trp(•)). In Escherichia coli photolyase, photoreduction of the flavin adenine dinucleotide (FAD) cofactor in its neutral radical semiquinone form (FADH(•)) results in the formation of FADH(-) and (306)Trp(•). Charge recombination between these two intermediates requires the uptake of a proton by (306)Trp(•). The rate constant of charge recombination has been measured as a function of temperature in the pH range from 5.5 to 10.0, and the data are analyzed with both classical Marcus and semi-classical Hopfield electron transfer theory. The reorganization energy associated with the charge recombination process shows a pH dependence ranging from 2.3 eV at pH ≤ 7 and 1.2 eV at pH(D) 10.0. These findings indicate that at least two mechanisms are involved in the charge recombination reaction. Global analysis of the data supports the hypothesis that PCET during charge recombination can follow two different mechanisms with an apparent switch around pH 6.5. At lower pH, concerted electron proton transfer (CEPT) is the favorable mechanism with a reorganization energy of 2.1-2.3 eV. At higher pH, a sequential mechanism becomes dominant with rate-limiting electron-transfer followed by proton uptake which has a reorganization energy of 1.0-1.3 eV. The observed 'inverse' deuterium isotope effect at pH < 8 can be explained by a solvent isotope effect that affects the free energy change of the reaction and masks the normal, mass-related kinetic isotope effect that is expected for a CEPT mechanism. To the best of our knowledge, this is the first time that a switch in PCET mechanism has been observed in a protein.

Analysis of measured and calculated Raman spectra of indole, 3-methylindole, and tryptophan on the basis of observed and predicted isotope shifts.

The aromatic amino acid tryptophan plays an important role in protein electron-transfer and in enzyme catalysis. Tryptophan is also used as a probe of its local protein environment and of dynamic changes in this environment. Raman spectroscopy of tryptophan has been an important tool to monitor tryptophan, its radicals, and its protein environment. The proper interpretation of the Raman spectra requires not only the correct assignment of Raman bands to vibrational normal modes but also the correct identification of the Raman bands in the spectrum. A significant amount of experimental and computational work has been devoted to this problem, but inconsistencies still persist. In this work, the Raman spectra of indole, 3-methylindole (3MI), tryptophan, and several of their isotopomers have been measured to determine the isotope shifts of the Raman bands. Density functional theory calculations with the B3LYP functional and the 6-311+G(d,p) basis set have been performed on indole, 3MI, 3-ethylindole (3EI), and several of their isotopomers to predict isotope shifts of the vibrational normal modes. Comparison of the observed and predicted isotope shifts results in a consistent assignment of Raman bands to vibrational normal modes that can be used for both assignment and identification of the Raman bands. For correct assignments, it is important to determine force field scaling factors for each molecule separately, and scaling factors of 0.9824, 0.9843, and 0.9857 are determined for indole, 3MI, and 3EI, respectively. It is also important to use more than one parameter to assign vibrational normal modes to Raman bands, for example, the inclusion of isotope shifts other than those obtained from H/D-exchange. Finally, the results indicate that the Fermi doublet of indole may consist of just two fundamentals, whereas one fundamental and one combination band are identified for the Fermi resonance that gives rise to the doublet in 3MI and tryptophan.

Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase-peroxidase (KatG).

The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.

Effect of the cyclobutane cytidine dimer on the properties of Escherichia coli DNA photolyase.

Cyclobutane pyrimidine dimer (CPD) photolyases are structure specific DNA-repair enzymes that specialize in the repair of CPDs, the major photoproducts that are formed upon irradiation of DNA with ultraviolet light. The purified enzyme binds a flavin adenine dinucleotide (FAD), which is in the neutral radical semiquinone (FADH(*)) form. The CPDs are repaired by a light-driven, electron transfer from the anionic hydroquinone (FADH(-)) singlet excited state to the CPD, which is followed by reductive cleavage of the cyclobutane ring and subsequent monomerization of the pyrimidine bases. CPDs formed between two adjacent thymidine bases (T< >T) are repaired with greater efficiency than those formed between two adjacent cytidine bases (C< >C). In this paper, we investigate the changes in Escherichia coli photolyase that are induced upon binding to DNA containing C< >C lesions using resonance Raman, UV-vis absorption, and transient absorption spectroscopies, spectroelectrochemistry, and computational chemistry. The binding of photolyase to a C< >C lesion modifies the energy levels of FADH(*), the rate of charge recombination between FADH(-) and Trp(306)(*), and protein-FADH(*) interactions differently than binding to a T< >T lesion. However, the reduction potential of the FADH(-)/FADH(*) couple is modified in the same way with both substrates. Our calculations show that the permanent electric dipole moment of C< >C is stronger (12.1 D) and oriented differently than that of T< >T (8.7 D). The possible role of the electric dipole moment of the CPD in modifying the physicochemical properties of photolyase as well as in affecting CPD repair will be discussed.

Contributions of the 8-methyl group to the vibrational normal modes of flavin mononucleotide and its 5-methyl semiquinone radical.

Resonance Raman spectroscopy is a powerful tool to investigate flavins and flavoproteins, and a good understanding of the flavin vibrational normal modes is essential for the interpretation of the Raman spectra. Isotopic labeling is the most effective tool for the assignment of vibrational normal modes, but such studies have been limited to labeling of rings II and III of the flavin isoalloxazine ring. In this paper, we report the resonance and pre-resonance Raman spectra of flavin mononucleotide (FMN) and its N5-methyl neutral radical semiquinone (5-CH 3FMN(*)), of which the 8-methyl group of ring I has been deuterated. The experiments indicate that the Raman bands in the low-frequency region are the most sensitive to 8-methyl deuteration. Density functional theory (DFT) calculations have been performed on lumiflavin to predict the isotope shifts, which are used to assign the calculated normal modes to the Raman bands of FMN. A first assignment of the low-frequency Raman bands on the basis of isotope shifts is proposed. Partial deuteration of the 8-methyl group reveals that the changes in the Raman spectra do not always occur gradually. These observations are reproduced by the DFT calculations, which provide detailed insight into the underlying modifications of the normal modes that are responsible for the changes in the Raman spectra. Two types of isotopic shift patterns are observed: either the frequency of the normal mode but not its composition changes or the composition of the normal mode changes, which then appears at a new frequency. The DFT calculations also reveal that the effect of H/D-exchange in the 8-methyl group on the composition of the vibrational normal modes is affected by the position of the exchanged hydrogen, i.e., whether it is in or out of the isoalloxazine plane.

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct.

Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.

A Review of Spectroscopic and Biophysical-Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA.

Cyclobutane pyrimidine dimer (CPD) photolyase (PL) is a structure-specific DNA repair enzyme that uses blue light to repair CPD on DNA. Cryptochrome (CRY) DASH enzymes use blue light for the repair of CPD lesions on single-stranded (ss) DNA, although some may also repair these lesions on double-stranded (ds) DNA. In addition, CRY DASH may be involved in blue light signaling, similar to cryptochromes. The focus of this review is on spectroscopic and biophysical-chemical experiments of the enzyme-substrate complex that have contributed to a more detailed understanding of all the aspects of the CPD repair mechanism of CPD photolyase and CRY DASH. This will be performed in the backdrop of the available X-ray crystal structures of these enzymes bound to a CPD-like lesion. These structures helped to confirm conclusions that were drawn earlier from spectroscopic and biophysical-chemical experiments, and they have a critical function as a framework to design new experiments and to interpret new experimental data. This review will show the important synergy between X-ray crystallography and spectroscopic/biophysical-chemical investigations that is essential to obtain a sufficiently detailed picture of the overall mechanism of CPD photolyases and CRY DASH proteins.

Resonance Raman spectroscopy of Compound II and its decay in Mycobacterium tuberculosis catalase-peroxidase KatG and its isoniazid resistant mutant S315T.

The reaction of Mycobacterium tuberculosis KatG and the mutant KatG(S315T) with two different organic peroxides is studied using resonance Raman spectroscopy. For the first time, an intermediate is observed in a catalase-peroxidase with vibrations that are characteristic of Compound II. The observation of this intermediate is consistent with photoreduction of Compound I and is in agreement with the formation of Compound I during the catalytic cycle of KatG. The same intermediate is detected in KatG(S315T), a mutant associated with resistance to isoniazid (INH), but with a lower yield, indicating that the organic peroxides cannot react with the heme iron in KatG(S315T) as efficiently as in wild-type KatG. Our results are consistent with catalytic competence of the S315T mutant and support the model that the S315T mutation confers antibiotic resistance by modifying the interaction between the enzyme and the drug.

Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae cryptochrome 1.

Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH(-)/FADH* reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH(-)/FADH* reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (K(A) approximately 10(5) M(-1) for photolyase and approximately 10(4) M(-1) for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed.

Antibiotic resistance in Mycobacterium tuberculosis: peroxidase intermediate bypass causes poor isoniazid activation by the S315G mutant of M. tuberculosis catalase-peroxidase (KatG).

KatG (catalase-peroxidase) in Mycobacterium tuberculosis is responsible for activation of isoniazid (INH), a pro-drug used to treat tuberculosis infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. The origin of INH resistance caused by the KatG[S315G] mutant enzyme is examined here. Overexpressed KatG[S315G] was characterized by optical, EPR, and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. Catalase activity and peroxidase activity with artificial substrates were moderately reduced (50 and 35%, respectively), whereas the rates of formation of oxyferryl heme:porphyrin pi-cation radical and the decay of heme intermediates were approximately 2-fold faster in KatG[S315G] compared with WT enzyme. The INH binding affinity for the resting enzyme was unchanged, whereas INH activation, measured by the rate of formation of an acyl-nicotinamide adenine dinucleotide adduct considered to be a bactericidal molecule, was reduced by 30% compared with WT KatG. INH resistance is suggested to arise from a redirection of catalytic intermediates into nonproductive reactions that interfere with oxidation of INH. In the resting mutant enzyme, a rapid evolution of 5-c heme to 6-c species occurred in contrast with the behavior of WT KatG and KatG[S315T] and consistent with greater flexibility at the heme edge in the absence of the hydroxyl of residue 315. Insights into the effects of mutations at residue 315 on enzyme structure, peroxidation kinetics, and specific interactions with INH are presented.

Role of the oxyferrous heme intermediate and distal side adduct radical in the catalase activity of Mycobacterium tuberculosis KatG revealed by the W107F mutant.

Catalase-peroxidase (KatG) is essential in Mycobacterium tuberculosis for oxidative stress management and activation of the antitubercular pro-drug isoniazid. The role of a unique distal side adduct found in KatG enzymes, involving linked side chains of residues Met255, Tyr229, and Trp107 (MYW), in the unusual catalase activity of KatG is addressed here and in our companion paper (Suarez, J., Ranguelova, K., Jarzecki, A. A., Manzerova, J., Krymov, V., Zhao, X., Yu, S., Metlitsky, L., Gerfen, G. J., and Magliozzo, R. S. (2009) J. Biol. Chem. 284, in press). The KatG[W107F] mutant exhibited severely reduced catalase activity yet normal peroxidase activity, and as isolated contains more abundant 6-coordinate heme in high spin and low spin forms compared with the wild-type enzyme. Most interestingly, oxyferrous heme is also found in the purified enzyme. Oxyferrous KatG[W107F] was prepared by photolysis in air of the carbonyl enzyme or was generated using hydrogen peroxide decayed with a t1/2 of 2 days compared with 6 min for wild-type protein. The stability of oxyenyzme was modestly enhanced in KatG[Y229F] but was not affected in KatG[M255A]. Optical stopped-flow experiments showed rapid formation of Compound I in KatG[W107F] and facile formation of oxyferrous heme in the presence of micromolar hydrogen peroxide. An analysis of the relationships between catalase activity, stability of oxyferrous enzyme, and a proposed MYW adduct radical is presented. The loss of catalase function is assigned to the loss of the MYW adduct radical and structural changes that lead to greatly enhanced stability of oxyenzyme, an intermediate of the catalase cycle of native enzyme.

Resonance Raman spectroscopic investigation of the light-harvesting chromophore in escherichia coli photolyase and Vibrio cholerae cryptochrome-1.

Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr.

Substrate binding modulates the reduction potential of DNA photolyase.

The reduction potential of the (FADH-/FADH*) couple in DNA photolyase was measured, and the value was found to be significantly higher than the values estimated in the literature. In the absence of substrate, the enzyme has a reduction potential of 16 +/- 6 mV vs NHE. In the presence of excess substrate the reduction potential increases to 81 +/- 8 mV vs NHE. The increase in reduction potential has physiological relevance since it gives the catalytic state greater resistance to oxidation. This is the first measurement of a reduction potential for this class of DNA-repair enzymes and the larger family of blue-light photoreceptors.

Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function.

Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm(-)(1) in the ferrous forms of both the wild-type enzyme and KatG(S315T). The ferrous-CO complex of both enzymes exhibits nu(CO), nu(Fe-CO), and delta(Fe-C-O) vibrations at 1925, 525, and 586 cm(-)(1), respectively, indicating a positive electrostatic environment for the CO complex, which is probably weakly hydrogen-bonded to a distal residue. The CO geometry is nonlinear as indicated by the unusually high intensity of the Fe-C-O bending vibration. The nu(Fe(III)-NO) and delta(Fe(III)-N-O) vibrations are detected at 596 and 571 cm(-)(1), respectively, in the ferric forms of wild-type and mutant enzyme and are indicative of a nonlinear binding geometry in support of the CO data. Although the presence of INH does not affect the vibrational frequencies of the CO- and NO-bound forms of either enzyme, it seems to perturb slightly their Raman intensities. Our results suggest a minimal, if any, perturbation of the distal heme pocket in the S315T mutant. Instead, the S315T mutation seems to induce small changes in the KatG conformation/dynamics of the ligand access channel as indicated by CO rebinding kinetics in flash photolysis experiments. The implications of these findings for the catalytic mechanism and mechanism of INH resistance in KatG(S315T) are discussed.

Resonance Raman spectroscopy of the neutral radical Trp306 in DNA photolyase.

The resonance Raman spectrum of the tryptophan neutral radical in a protein, Escherichia coli photolyase, is reported for the first time. The data compare very well to a solution study and computational predictions, and tentative assignments are made for the observed vibrations. This important new result demonstrates the potential of time-resolved resonance Raman spectroscopy as a powerful tool to investigate these radicals in protein electron-transfer processes and in enzymatic reactions in real time.

Resonance Raman investigation of the interaction of thromboxane synthase with substrate analogues.

Thromboxane synthase is a hemethiolate enzyme that catalyzes the isomerization of prostaglandin H2 to thromboxane A2. We report the first resonance Raman (RR) spectra of recombinant human thromboxane synthase (TXAS) in both the presence and the absence of substrate analogues U44069 and U46619. The resting enzyme and its U44069 complex are found to have a 6-coordinate, low spin (6c/ls) heme, in agreement with earlier experiments. The U46619-bound enzyme is detected as a 6c/ls heme too, which is in contradiction with a previous conclusion based on absorption difference spectroscopy. Two new vibrations at 368 and 424 cm(-1) are observed upon binding of the substrate analogues in the heme pocket and are assigned to the second propionate and vinyl bending modes, respectively. We interpret the changes in these vibrational modes as the disruption of the protein environment and the hydrogen-bonding network of one of the propionate groups when the substrate analogues enter the heme pocket. We use carbocyclic thromboxane A2 (CTA2) to convert the TXAS heme cofactor to its 5-coordinate, high spin (5c/hs) form, as is confirmed by optical and RR spectroscopy. In this 5c/hs state of the enzyme, the Fe-S stretching frequency is determined at 350 cm(-1) with excitation at 356.4 nm. This assignment is supported by comparison to the spectrum of resting enzyme excited at 356.4 nm and by exciting at different wavelengths. Implications of our findings for substrate binding and the catalytic mechanism of TXAS will be discussed.

Resonance Raman detection of the Fe-S bond in endothelial nitric oxide synthase.

We report the first low-frequency resonance Raman spectra of ferric endothelial nitric oxide synthase (eNOS) holoenzyme, including the frequency of the Fe-S vibration in the presence of the substrate L-arginine. This is the first direct measurement of the strength of the Fe-S bond in NOS. The Fe-S vibration is observed at 338 cm(-1) with excitation at 363.8 nm. The assignment of this band to the Fe-S stretching vibration was confirmed by the observation of isotopic shifts in eNOS reconstituted with 54Fe- and 57Fe-labeled hemin. Furthermore, the frequency of this vibration is close to those observed in cytochrome P450(cam) and chloroperoxidase (CPO). The frequency of this vibration is lower in eNOS than in P450(cam) and CPO, which can be explained by differences in hydrogen bonding to the proximal cysteine heme ligand. On addition of substrate to eNOS, we also observe several low-frequency vibrations, which are associated with the heme pyrrole groups. The enhancement of these vibrations suggests that substrate binding results in protein-mediated changes of the heme geometry, which may provide the protein with an additional tuning element for the redox potential of the heme iron. The implications of our findings for the function of eNOS will be discussed by comparison with P450(cam) and model compounds.

Substrate electric dipole moment exerts a pH-dependent effect on electron transfer in Escherichia coli photolyase.

Transient absorption spectroscopy is used to demonstrate that the electric dipole moment of the substrate cyclobutane thymine dimer affects the charge recombination reaction between fully reduced flavin adenine dinucleotide (FADH-) and the neutral radical tryptophan 306 (Trp306*) in Escherichia coli DNA photolyase. At pH 7.4, the charge recombination is slowed by a factor of 1.75 in the presence of substrate, but not at pH 5.4. Photolyase does bind substrate at pH 5.4, and it seems that this pH effect originates from the conversion of FADH- to FADH2 at lower pH. The free-energy changes calculated from the electric field parameters and from the change in electron transfer rate are in good agreement and support the idea that the substrate electric dipole is responsible for the observed change in electron transfer rate. It is expected that the substrate electric field will also modify the physiologically important from excited 1FADH- to the substrate in the DNA repair reaction.