On the enigma of old yellow enzyme's spectral properties

Old yellow enzyme (NADPH oxidoreductase) in the free and complexed state was thoroughly investigated by the following techniques: absorption, circular dichroism, fluorescence/phosphorescence and electron paramagnetic resonance spectroscopy and fluorescence/phosphorescence decay measurements, applied over a wide range of temperature (7-293K). The data obtained were interpreted by comparison with results from similar measurements on free FMN, existing spectral data on isoalloxazine model systems and theoretical data. The results clearly demonstrate the inadequacy of a simple phenolate-FMN donor-acceptor charge-transfer complex to explain the phenomena occurring upon the addition of phenols to old yellow enzyme. Instead it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably an H-bonded structure in which FMN is tautomerized and interacts with an L-chiral center. This is concluded from a separate electronic transition with an origin at 496 nm, thus far not recognized as such, and the circular dichroism observed. The emission is dominated by that of free FMN, although protein-bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon excitation of both native and complexed old yellow enzyme in the ultraviolet. This emission is quenched by the addition of phenol to the enzyme, shows a large (3000-cm-1) blue shift on going to a low-temperature glass and is tentatively assigned to excimers of nucleic acids. Long-wavelength excitation with a synchronously pumped, mode-locked Rhodamine 6-G dye laser revealed a third, extremely weak emission in both native old yellow enzyme and its complexes. It decays with a lifetime of about 3 ns at 143 K. Electron paramagnetic resonance spectra revealed the presence of a low amount of an unpaired spin in old yellow enzyme. Owing to an unusual relaxational behaviour it could only be observed below 15 K and, again, the signal was measured in both the native enzyme and its complexes. Possible assignment and consequences of this observation are discussed. In frozen aqueous solutions of the enzyme-phenolate complex, a phase transition was discovered at which the colour of the complex reverted to that of the native enzyme. Subsequent melting restored the original colour. The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge is that of a very complicated network of hydrogen-bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long-wavelength absorption band in old yellow enzyme.     

Old yellow enzymes protect against acrolein toxicity in the yeast Saccharomyces cerevisiae

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce alpha,beta-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of alpha,beta-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small alpha,beta-unsaturated carbonyl compounds, such as acrolein, in vivo.     

Old Yellow Enzymes Protect against Acrolein Toxicity in the Yeast Saccharomyces cerevisiae

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce α,β-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of α,β-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small α,β-unsaturated carbonyl compounds, such as acrolein, in vivo.     

Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP.

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.     

Oxygen detoxification by dienoyl-CoA oxidase involving flavin/disulfide cofactors

Class I benzoyl-CoA reductases (BCRs) are oxygen-sensitive key enzymes in the degradation of monocyclic aromatic compounds in anaerobic prokaryotes. They catalyze the ATP-dependent reductive dearomatization of their substrate to cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA). An aromatizing 1,5-dienoyl-CoA oxidase (DCO) activity has been proposed to protect BCRs from oxidative damage, however, the gene and its product involved have not been identified, yet. Here, we heterologously produced a DCO from the hyperthermophilic euryarchaeon Ferroglobus placidus that coupled the oxidation of two 1,5-dienoyl-CoA to benzoyl-CoA to the reduction of O₂ to water at 80°C. DCO showed similarities to members of the old yellow enzyme family and contained FMN, FAD and an FeS cluster as cofactors. The O₂-dependent activation of inactive, reduced DCO is assigned to a redox thiol switch at E^o′ = −3 mV. We propose a catalytic cycle in which the active site FMN/disulfide redox centers are reduced by two 1,5-dienoyl-CoA (reductive half-cycle), followed by two consecutive two-electron transfer steps to molecular oxygen via peroxy- and hydroxyflavin intermediates yielding water (oxidative half-cycle). This work identified the enzyme involved in a unique oxygen detoxification process for an oxygen-sensitive catabolic enzyme.     

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.     

Covalently bound flavin as prosthetic group of choline oxidase.

Highly purified choline oxidase of $\text{Arthrobacter globiformis}$ fluoresced as a yellow band on SDS gel in 7% acetic acid. The absorption spectrum of the enzyme showed marked hypsochromic shift of the second absorption band. Aminoacyl flavin obtained from this enzyme was identified with 8α-[N(3)-histidyl]FAD.     

Identification and characterization of 2‐naphthoyl‐coenzyme A reductase,the prototype of a novel class of dearomatizing reductases

The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl‐coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl‐CoA by ATP‐dependent or ‐independent benzoyl‐CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2‐naphthoyl‐CoA reductase was purified from extracts of the naphthalene‐degrading, sulphidogenic enrichment culture N47. The oxygen‐tolerant enzyme dearomatized the non‐activated ring of 2‐naphthoyl‐CoA by a four‐electron reduction to 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin‐containing ‘old yellow enzyme’ family. NCR contained FAD, FMN, and an iron‐sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2‐naphthoyl‐CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl‐CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl‐CoA reductases.     

EPR spectroscopy of soybean lipoxygenase-1. Description and quantification of the high-spin fe(III) signals

The interaction of soybean lipoxygenase-1 with 13-Ls-hydroperoxy-9-cis,11-trans-octadecadienoic acid, the product of the enzymic dioxygenation of linoleic acid, results in the formation of either a yellow or a purple coloured enzyme form depending on the amount of product used. The composition of the high-spin Fe(III) signals in the EPR spectra of both enzyme forms has been studied and the amount of EPR-visible iron determined by integration and simulation. Sets of g values of the species building up the high-spin Fe(III) signal around g 6 are derived from both third-order perturbation calculation and exact numerical diagonalization of the spin Hamiltonian describing the system. The results of these calculations are generally applicable to systems having S = 5/2. The iron in the native, colourless enzyme is almost EPR-nondetectable. The yellow form of the enzyme shows a complex EPR signal around g 6 which consists of contributions of at least three species with different ligand symmetry. The signal corresponds to approx. 75% of the total iron content. The g 6 signal of the purple Fe(III) enzyme corresponds roughly to the same amount of iron but the ratio between the different species is not the same as in the yellow enzyme. This enzyme form also shows an additional g 4.3 signal with a large amplitude but a relatively low integrated intensity (approx. 10% of the total iron content). The results are consistent with the suggested mechanism of the catalytic function of iron in lipoxygenase which was based on qualitative EPR results (De Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J., Wever, R. and van Gelder, B.F. (1975) Biochim. Biophys. Acta 377, 71--79).     

Aromatic side-chain cluster of biotin binding site of avidin allows circular dichroism spectroscopic investigation of its ligand binding properties

Promiscuous ligand binding by hen egg-white avidin has been demonstrated and studied by using circular dichroism (CD) spectroscopy complemented by molecular docking calculations. It has been shown that the biotin-binding pocket of avidin is able to accommodate a wide variety of chemical compounds including therapeutic drugs (e.g., thalidomide, NSAIDs, antihistamines), natural compounds (bilirubin, myristic acid), and synthetic agents (xanthenone dyes). The cluster of aromatic residues located at the biotin-binding pocket renders the intrinsic CD spectrum of avidin sensitive to ligand binding that results in the increase of the vibronic components of the (1) L(b) transition of the Trp residues. Extrinsic (induced) CD bands measured with chemically diverse avidin ligands are generated by intramolecular coupled oscillator (e.g., bilirubin) or by intermolecular ligand-Trp exciton coupling mechanism [e.g., 2-(4'-hydroxyazobenzene)-benzoic acid (HABA)]. Among the compounds of which avidin-binding affinity constants have been calculated, two novel high-affinity ligands, flufenamic acid and an enzyme inhibitor thiazole derivative have been identified (K(d) ≈ 1 μM). Avidin binding mode of the ligand molecules has been discussed in the light of docking results. The induced CD profile of the thiazole derivative has been correlated with the stereochemistry of its docked conformation. The important role in the ligand binding of a polar side-chain cluster at the bottom of the biotin-binding cavity as well as the analogous avidin-binding mode of HABA and fenamic acid type NSAIDs have been proposed.     

Measurement of the electronic properties of the flavoprotein old yellow enzyme (OYE) and the OYE:p-Cl phenol charge-transfer complex using Stark spectroscopy

Low-temperature absorption and Stark spectroscopy have been used to study the electronic properties of oxidized flavin mononucleotide (FMN) in old yellow enzyme (OYE) and OYE complexed with p-chlorophenol (p-Cl phenol). The low-temperature absorbance spectrum of OYE showed splittings of the blue and near-UV vibronic bands, which appears to be due to hydrogen bonding between the isoalloxazine moiety and the protein. A Stark spectroscopic analysis showed that the electronic structure of the FMN cofactor in OYE is not significantly perturbed relative to flavins in simple solvents. However, the charge-transfer band in the OYE:p-Cl phenol complex showed a large Stark effect indicative of substantial charge displacement. The magnitude and direction of this charge displacement are consistent with significant charge transfer along the charge-transfer transition dipole moment direction. In addition, the Stark spectrum of the CT band showed unexpected fine structure that could correlate with vibrational progressions in either the p-Cl phenol donor or the flavin acceptor.     

The purification and properties of butyryl-coenzyme A dehydrogenase from Peptostreptococcus elsdenii

Butyryl-CoA dehydrogenase prepared by a simple procedure from Peptostreptococcus elsdenii has a molecular weight of approx. 150000. The enzyme has FAD as its prosthetic group. The amino acid analysis is reported. This enzyme, like most of the corresponding mammalian ones, is green. The absorption band at 710nm can be abolished irreversibly by dithionite reduction and air reoxidation; it can be abolished reversibly by phenylmercuric acetate or potassium bromide. The enzyme as isolated appears to be a mixture of a green and a yellow form, both of which are active. This view is supported by the variable ;greenness' of different preparations and the biphasic curve obtained in anaerobic spectrophotometric titrations with dithionite. It can be calculated from the titration results that fully green enzyme would have a peak-to-peak absorption ratio (E(710)/E(430)) as great as 0.54. The green form is much less rapidly reduced by dithionite than the yellow form, but is nevertheless much more readily reduced by dithionite than the enzyme from pig liver. It is also more readily reoxidized by air and shows less tendency to form a semiquinone. Treatment with sodium borohydride produces an unusual reduced species that is probably the 3,4-dihydroflavin.     

Procatalytic ligand strain. Ionization and perturbation of 8-nitroxanthine at the urate oxidase active site

The binding of the inhibitor 8-nitroxanthine to urate oxidase has been investigated by Raman and UV-visible absorption spectroscopy. The absorption maximum of 8-nitroxanthine shifts from 380 to 400 nm upon binding to the enzyme, demonstrating that the electronic structure of the ligand is perturbed. It has been proposed that oxidation of the substrate urate by urate oxidase is facilitated by formation of the substrate dianion at the enzyme active site, and Raman spectra of urate oxidase-bound 8-nitroxanthine suggest that both the dianionic and monoanionic forms of the ligand are bound to the enzyme under conditions where in solution the monoanion is present exclusively. The C4-C5 stretching frequency appears as a relatively isolated vibrational mode in 8-nitroxanthine whose frequency shifts according to the protonation state of the purine ring. Identification of the C4-C5 stretching mode was confirmed using [4-(13)C]-8-nitroxanthine and ab initio calculation of the vibrational modes. Two peaks corresponding to the C4-C5 stretching mode were evident in spectra of enzyme-bound 8-nitroxanthine, at 1541 and 1486 cm(-)(1). The higher frequency peak was assigned to monoanionic 8-nitroxanthine, and the low-frequency peak was assigned to dianionic 8-nitroxanthine. The C4-C5 stretching frequency for free monoanionic 8-nitroxanthine was at 1545 cm(-)(1), indicating that the enzyme polarizes that bond when the ligand is bound. The C4-C5 stretching frequency in dianionic 8-nitroxanthine is also shifted by 4 cm(-)(1) to lower frequency upon binding. For 8-nitroxanthine free in solution, the C4-C5 stretching frequency shifts to lower frequency upon deprotonation, and the absorption maximum in the UV-visible spectrum shifts to higher wavelength. The spectral shifts observed upon binding of 8-nitroxanthine to urate oxidase are consistent with increased anionic character of the ligand, which is expected to promote catalysis in the reaction with the natural substrate urate. In the Raman spectra of 8-nitroxanthine bound to the F179A, F179Y, and K9M mutant proteins, the C4-C5 stretching frequency was not perturbed from its position for the unbound ligand. Both V(max) and V/K were decreased in the mutant enzymes, demonstrating a correlation between the interaction that perturbs the C4-C5 stretching frequency and the catalytic activity of the enzyme. It is suggested that hydrogen-bonding interactions that lead to precise positioning and deprotonation of the substrate are perturbed by the mutations.     

Two gossypetin methyl ethers as ultraviolet patterning guides in the flowers of Coronilla valentina

The 3′-monomethyl and 8,3′-dimethyl ethers of gossypetin have been identified in the flowers of Coronilla valentine where they occur as the 3-rutinosides. These two yellow flavonols occur specifically in the wings and thus provide both visible yellow colour and UV absorption to bees, which land on the wings and trigger the self-fertilization mechanism. These yellow pigments are absent from the flowers of the related C. emerus, where their role in UV patterning is taken over by colourless kaempferol and quercetin glycosides.     

Ligand-induced conformational changes in the capping subdomain of a bacterial old yellow enzyme homologue and conserved sequence fingerprints provide new insights into substrate binding

We have recently reported that Shewanella oneidensis, a Gram-negative gamma-proteobacterium with a rich arsenal of redox proteins, possesses four old yellow enzyme (OYE) homologues. Here, we report a series of high resolution crystal structures for one of these OYEs, Shewanella yellow enzyme 1 (SYE1), in its oxidized form at 1.4A resolution, which binds a molecule of PEG 400 in the active site, and in its NADH-reduced and p-hydroxybenzaldehyde- and p-hydroxyacetophenone-bound forms at 1.7A resolution. Although the overall structure of SYE1 reveals a monomeric enzyme based on the alpha(8)beta(8) barrel scaffold observed for other OYEs, the active site exhibits a unique combination of features: a strongly butterfly-bent FMN cofactor both in the oxidized and NADH-reduced forms, a collapsed and narrow active site tunnel, and a novel combination of conserved residues involved in the binding of phenolic ligands. Furthermore, we identify a second p-hydroxybenzaldehyde-binding site in a hydrophobic cleft next to the entry of the active site tunnel in the capping subdomain, formed by a restructuring of Loop 3 to an "open" conformation. This constitutes the first evidence to date for the entire family of OYEs that Loop 3 may indeed play a dynamic role in ligand binding and thus provides insights into the elusive NADH complex and into substrate binding in general. Structure-based sequence alignments indicate that the novelties we observe in SYE1 are supported by conserved residues in a number of structurally uncharacterized OYEs from the beta- and gamma-proteobacteria, suggesting that SYE1 represents a new subfamily of bacterial OYEs.     

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

Determinants of Substrate Binding and Protonation in the Flavoenzyme Xenobiotic Reductase A

Xenobiotic reductase A (XenA) from Pseudomonas putida 86 catalyzes the NAD(P)H-dependent reduction of various α,β-unsaturated carbonyl compounds and is a member of the old yellow enzyme family. The reaction of XenA follows a ping-pong mechanism, implying that its active site has to accommodate and correctly position the various substrates to be oxidized (NADH/NADPH) and to be reduced (different α,β-unsaturated carbonyl compounds) to enable formal hydride transfers between the substrate and the isoalloxazine ring.The active site of XenA is lined by two tyrosine (Tyr27, Tyr183) and two tryptophan (Trp302, Trp358) residues, which were proposed to contribute to substrate binding. We analyzed the individual contributions of the four residues, using site-directed mutagenesis, steady-state and transient kinetics, redox potentiometry and crystal structure analysis. The Y183F substitution decreases the affinity of XenA for NADPH and reduces the rate of the oxidative half-reaction by two to three orders of magnitude, the latter being in agreement with its function as a proton donor in the oxidative half-reaction. Upon reduction of the flavin, Trp302 swings into the active site of XenA (in-conformation) and decreases the extent of the substrate-binding pocket. Its exchange against alanine induces substrate inhibition at elevated NADPH concentrations, indicating that the in-conformation of Trp302 helps to disfavor the nonproductive NADPH binding in the reduced state of XenA.Our analysis shows that while the principal catalytic mechanism of XenA, for example, type of proton donor, is analogous to that of other members of the old yellow enzyme family, its strategy to correctly position and accommodate different substrates is unprecedented.     

A light-sensitive yellow pigment from the housefly

Extraction of house-fly heads with neutral phosphate buffer yielded a dark brown solution from which a number of pigments were separated, either wholly or partially, by chromatography on a column of calcium phosphate mixed with celite. One of the pigments was light-sensitive, and had a yellow color, with a spectral absorption maximum at 437 mmicro in phosphate buffer at pH 6.5. Several consecutively eluted fractions from each chromatogram of the house-fly head extract showed the characteristic absorption curve of this pigment with no trace, spectroscopically, of the other pigments of the extract. The products of bleaching the pigment at pH 6.5 had an absorption curve showing plateaus at 440 to 460 mmicro and 350 to 360 mmicro and an inflection at about 250 mmicro. Above pH 8.0 the pigment bleached in the dark giving an absorption maximum at about 380 mmicro, and inflections at 290 mmicro and at about 250 mmicro. With 2.5 to 5 N HCl or H(2)SO(4) an absorption maximum at 470 to 475 mmicro was obtained with either the unbleached or the bleached pigment. With sulfosalicylic acid, ethanol, or heating at 100 degrees C., a part of the pigment was precipitated, leaving a light-stable yellow supernatant. This light-sensitive house-fly pigment cannot as yet be identified with any of the previously known insect pigments or with the photosensitive pigments of other animals, though these latter compounds exhibit some similarity in their spectroscopic properties.     

Hydrophobic interaction of alpha-chymotrypsin with low molecular weight compounds

Data on alpha-chymotrypsin interactions with hydrophobic low-molecular compounds have been generalized. Existence of two sites of noncovalent interaction with hydrophobic nuclei of a ligand molecule is shown. When the substance to be bound contains only one hydrophobic nucleus, the interaction is mediated by a "hydrophobic pocket" of the enzyme--a binding site of amino acid residues which are, in the P1-position relative to the cleaved bond. Under these conditions substances with an asymmetric hydrophobic nucleus (of the tryptophan type) are better ligands for binding. In case of compounds containing several hydrophobic groups scattered in the space, interaction with the enzyme proceeds in two binding sites. New data are presented on the ligand specificity for binding sites of chymotrypsin in lower vertebrates. Relative position of hydrophobic groups of the ligand is shown as that of great importance for interaction with the enzyme. It is concluded that the binding sites of trypsin- and chymotrypsin-like proteinases of the lower vertebrates differ but less from each other as compared to binding sites of trypsin and chymotrypsin in mammals.