Differences between the electric fields of the catalytic sites of papain and actinidin detected by using the thiol-located nitrobenzofurazan label as a spectroscopic reporter group.

The catalytic-site thiol groups of papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were each labelled with the nitrobenzofurazan (Nbf) chromophore by reaction with 4-chloro-7-nitrobenzofurazan at pH 4.4. The electronic-absorption spectra of both labelled enzymes were determined in aqueous solution, in the pH ranges approx. 2-5 for S-Nbf-papain and approx. 3.3-8 for S-Nbf-actinidin, and for the latter also in 6 M-guanidinium chloride. The spectrum of S-Nbf-papain is characterized by lambda max. = 402 nm at pH 5 and by lambda max. = 422 nm at pH 2.18. The pH-dependent shift in lambda max. accompanies a pH-dependent change in A 430, the nature of which is consistent with its dependence on a single ionizing group with pKa 3.7. The spectrum of S-Nbf-actinidin is pH-independent in the pH range approx. 3.3-8 and is characterized by lambda max. = 413 nm. This absorption maximum shifts to 425 nm in 6M-guanidinium chloride. These results are discussed and related to those reported previously from studies on papain and actinidin with various reactivity probes. Despite the close similarity in the catalytic sites of papain and actinidin deduced from X-ray-diffraction studies, the considerable differences in their reactivity characteristics are mirrored by differences in their electric fields detected by the Nbf spectroscopic label. The microenvironment in the catalytic site of actinidin appears to favour the existence of ions significantly more than in the corresponding region in papain.     

Spectral characteristics of phytochrome in vivo and in vitro

The absorption maximum of the far-red absorbing form of phytochrome in the difference spectrum for phototransformation (Pfr max) was investigated in vivo and in in vitro pellets from dark grown Hordeum vulgare L. primary leaves. Exposure of pellets in Honda medium from tissue pre-irradiated with red light to far red light gave a Pfr max of 734 nm, a slightly longer wavelength than was seen in vivo (730 nm). After incubation as the red absorbing form of phytochrome (Pr) for 2 h at 0° C irradiation with red light showed that Pfr max had shifted to shorter wavelength (716 nm) in Honda medium. Further incubation as Pfr for 2 h at 0° C and irradiation with far red light showed that Pfr max had shifted to longer wavelength (726 nm). Similar shifts were also seen in other media, although the peak positions were different. Phytochrome remained pelletable throughout these experiments and Pfr max is compared to that of soluble phytochrome in similar media. The results are interpreted as indicating changes in molecular environment of the putative phytochrome membrane receptor site and that Pfr max can be used to probe the nature of this binding.Abbreviations D Dark - EDTA Ethylene diamine tetra-acetic acid - F far red light - MOPS N-morpholino-3-propane-sulphonic acid - P Phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - Pfr max wavelength maximum of Pfr absorbance in a phototransformation difference spectrum - R red light     

Comparative study of the structural characteristics of aldolase in rabbit muscles in normal states and in alloxan diabetes

It is shown that the activity of aldolase synthesized in rabbit muscles under diabetes is higher than that at normal state. This fact is probably a result of some structural alterations in NAD-binding site with Trp-291 and -311 in it which overlaps a considerable part of C-terminal region of the protein. The hydrophobic part of the enzyme containing Trp-147 under diabetes seems to remain unaltered. This consideration is based on the longwave shift in aldolase fluorescence lambda max (from 320 to 324 nm) under this pathology, suggesting a transition of Trp-291 and -311 into more polar environment and is confirmed by the disappearance of the difference in lambda max in the NADH presence. The NADH-originated shift in lambda max position for the both proteins ended at the same wave-length at 314 nm. The position of lambda max at 324 nm resulting from possible structural modification of NAD-binding site under diabetes correlates with an increase in the Stern-Volmer quenching constant value (from 4359 to 7500 M-1 for aldolase under normal and diabetic states, respectively). These quenching data evidence in favour of the suggestion on the existence of two classes of tryptophanyls in the aldolase molecule.     

Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies [Lane, A., & Kirschner, K. (1983) Eur. J. Biochem. 129, 561-570], the reaction with alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3). The RSSF data reveal that during tau 1, the internal aldimine, E(PLP), with lambda max = 412 nm (pH 7.8), undergoes rapid conversion to two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process (1/tau 2, 1/tau 3) to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in tau 2 are nearly identical with the spectral changes in tau 3. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with lambda max congruent to 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the alpha proton from the external aldimine. The reaction of L-serine with beta 2 consists of two relaxations (1/tau 1 beta greater than 1/tau 2 beta) and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report [Miles, E. W., Hatanaka, M., & Crawford, I. P. (1968) Biochemistry 7, 2742-2753]. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in tau 2 and tau 3 of the alpha 2 beta 2 reaction is postulated to originate from the existence of two (slowly) interconverting forms of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of fructose-1,6-bisphosphatase by o-phthalaldehyde

Rabbit liver fructose-1,6-bisphosphatase, a tetramer of identical subunits was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The second-order rate constant for the inactivation was 30 M-1s-1. Fructose-1,6-bisphosphatase was completely protected from inactivation by the substrate--fructose-1,6-diphosphate but not by the allosteric effector--adenosine monophosphate. The absorption spectrum (lambda max 337 nm) and, fluorescence excitation (lambda max 360 nm) and fluorescence emission spectra (lambda max 405 nm) were consistent with the formation of an isoindole derivative in the subunit between a cysteine and a lysine residue about 3A apart. About 4 isoindole groups per mol of the bisphosphatase were formed following complete loss of the phosphatase activity. This suggests that the amino acid residues of the biphosphatase participating in reaction with o-phthalaldehyde more likely reside at or near the active site instead of allosteric site. The molar transition energy of fructose-1,6-bisphosphatase--o-phthalaldehyde adduct was estimated 121 kJ/mol and compares favorably with 127 kJ/mol for the synthetic isoindole, 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl) isoindole in hexane. It is, thus, concluded that the cysteine and lysine residues participating in isoindole formation in reaction between fructose-1,6-bisphosphatase and o-phthalaldehyde are located in a hydrophobic environment.     

Spectral tuning of avian violet- and ultraviolet-sensitive visual pigments

The violet- and ultraviolet-sensitive visual pigments of birds belong to the same class of pigments as the violet-sensitive (so-called blue) pigments of mammals. However, unlike the pigments from mammals and other vertebrate taxa which, depending on species, have lambda(max) values of either around 430 nm or around 370 nm, avian pigments are found with lambda(max) values spread across this range. In this paper, we present the sequences of two pigments isolated from Humbolt penguin and pigeon with intermediate lambda(max) values of 403 and 409 nm, respectively. By comparing the amino acid sequences of these pigments with the true UV pigments of budgerigar and canary and with chicken violet with a lambda(max) value of 420 nm, we have been able to identify five amino acid sites that show a pattern of substitution between species that is consistent with differences in lambda(max). Each of these substitutions has been introduced into budgerigar cDNA and expressed in vitro in COS-7 cells. Only three resulted in spectral shifts in the regenerated pigment; two had relatively small effects and may account for the spectral shifts between penguin, pigeon, and chicken whereas one, the replacement of Ser by Cys at site 90 in the UV pigments, produced a 35 nm shortwave shift that could account for the spectral shift from 403 nm in penguin to around 370 nm in budgerigar and canary.     

Specific binding sites for cations in bacteriorhodopsin.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn(2+) and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn(2+), which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be approximately 9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn(2+) bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.     

Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.     

Variations of colour vision in a New World primate can be explained by polymorphism of retinal photopigments

The squirrel monkey (Saimiri sciureus) exhibits a polymorphism of colour vision: some animals are dichromatic, some trichromatic, and within each of these classes there are subtypes that resemble the protan and deutan variants of human colour vision. For each of ten individual monkeys we have obtained (i) behavioural measurements of colour vision and (ii) microspectrophotometric measurements of retinal photopigments. The behavioural tests, carried out in Santa Barbara, included wavelength discrimination, Rayleigh matches, and increment sensitivity at 540 and 640 nm. The microspectrophotometric measurements were made in London, using samples of fresh retinal tissue and a modified Liebman microspectrophotometer: the absorbance spectra for single retinal cells were obtained by passing a monochromatic measuring beam through the outer segments of individual rods and cones. The two types of data, behavioural and microspectrophotometric, were obtained independently and were handed to a third party before being interchanged between experimenters. From all ten animals, a rod pigment was recorded with lambda max (wavelength of peak absorbance) close to 500 nm. In several animals, receptors were found that contained a short-wave pigment (mean lambda max = 433.5 nm): these violet-sensitive receptors were rare, as in man and other primate species. In the middle- to long-wave part of the spectrum, there appear to be at least three possible Saimiri photopigments (with lambda max values at about 537,550 and 565 nm) and individual animals draw either one or two pigments from this set, giving dichromatic or trichromatic colour vision. Thus, those animals that behaviourally resembled human protanopes exhibited only one pigment in the red-green range, with lambda max = 537 nm; other behaviourally dichromatic animals had single pigments lying at longer wavelengths and these were the animals that behaviourally had higher sensitivity to long wavelengths. Four of the monkeys were behaviourally judged to be trichromatic. None of the latter animals exhibited the two widely separated pigments (close to 535 and 567 nm) that are found in the middle- and long-wave cones of macaque monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)     

Absolute absorption spectra of batho- and photorhodopsins at room temperature. Picosecond laser photolysis of rhodopsin in polyacrylamide.

Picosecond laser photolysis of rhodopsin in 15% polyacrylamide gel was performed for estimating absolute absorption spectra of the primary intermediates of cattle rhodopsin (bathorhodopsin and photorhodopsin). Using a rhodopsin digitonin extract embedded in 15% polyacrylamide gel, a precise percentage of bleaching of rhodopsin after excitation of a picosecond laser pulse was measured. Using this value, the absolute absorption spectrum of bathorhodopsin was calculated from the spectral change before and 1 ns after the picosecond laser excitation (corresponding to the difference spectrum between rhodopsin and bathorhodopsin). The absorption spectrum of bathorhodopsin thus obtained displayed a lambda max at 535 nm, which was shorter than that at low temperature (543 nm) and a half band-width broader than that measured at low temperature. The oscillator strength of bathorhodopsin at room temperature was smaller than that at low temperature. The absolute absorption spectrum of photorhodopsin was also estimated from the difference spectrum measured at 15 ps after the excitation of rhodopsin (Shichida, Y., S. Matuoka, and T. Yoshizawa. 1984. Photobiochem. Photobiophys. 7:221-228), assuming a sequential conversion of photorhodopsin to bathorhodopsin. Its lambda max was located at approximately 570 nm, and the oscillator strength was smaller than those of rhodopsin and bathorhodopsin.     

A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts

A method for measuring the activity of the pyruvate dehydrogenase complex (PDC) by coupling acetyl-CoA production to acetylation of a fluorescent dye is described. Acetylation of cresyl violet acetate by pigeon liver acetyltransferase results in a shift of its fluorescence spectrum from lambda ex max = 575, lambda em max = 620 nm to lambda ex max = 475, lambda em max = 575 nm. The rate of appearance of acetylated dye was followed fluorometrically and was proportional to PDC activity in extracts of cultured human fibroblasts. The assay showed appropriate substrate and cofactor dependence and had a working range between 0.04 and 70 munits. It is 10 times more sensitive than the spectrophotometric assay on which it is based (working range 0.4-31 munits) and is equally convenient. Unactivated PDC activity in fibroblast extracts was 0.75 (0.60-0.92) munits/mg protein (mean and range for six cell lines).     

Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.     

Kinetics of the release of aluminum from human serum dialuminum transferrin to citrate

The kinetics of release of Al3+ from human serum dialuminum transferrin (Al2Tf) to citrate were investigated at 37 degrees C, pH 7.4, mu = 0.7 M, by difference UV spectrophotometry. The two metal-binding sites are not identical but behave in a kinetically similar manner to give apparent second-order rate constants of 0.60 and 0.38 M-1 s-1, respectively, for release of the first Al3+ from Al2Tf. The rate constants for release of the second metal ion from the monoaluminum transferrins are 0.27 and 0.12 M-1 s-1. The kinetic scheme for release of A13+ from Al2Tf is therefore similar to that for release of Fe3+ from Fe2Tf, but the rate of constants for metal ion release are between two and four orders of magnitude larger.     

4-Hydroxycinnamoyl-CoA: an ionizable probe of the active site of the medium chain acyl-CoA dehydrogenase

4-OH-Cinnamoyl-CoA has been synthesized as a probe of the active site in the medium chain acyl-CoA dehydrogenase. The protonated form of the free ligand (lambda(max) = 336 nm) yields the corresponding phenolate (lambda(max) = 388 nm) with a pK of 8.9. 4-OH-Cinnamoyl-CoA binds tightly (K(d) = 47 nM, pH 6) to the pig kidney dehydrogenase with a prominent new band at 388 nm, suggesting ionization of the bound ligand. However, this spectrum reflects polarization, not deprotonation, of the neutral form of the ligand. Thus, the 388 nm band is abolished as the pH is raised (not lowered), and analogous spectral and pH behavior is observed with the nonionizable analogue 4-methoxycinnamoyl-CoA. Studies with wild type, E99G, and E376Q mutants of the human medium chain acyl-CoA dehydrogenase showed that these two active site carboxylates strongly suppress ionization of the 4-OH ligand. Binding to the double mutant E99G/E376Q gives an intense new band as the pH is raised (pK = 7.8), with an absorbance maximum at 498 nm resembling the natural 4-OH-cinnamoyl-thioester chromophore of the photoactive yellow protein. Raman difference spectroscopy in water and D(2)O, using the free ligand and wild-type and double-mutant enzyme.ligand complexes, confirms that the 4-OH group of the thioester is ionized only when bound to the double mutant. These data demonstrate the strong electrostatic coupling between ligand and enzyme, and the critical role Glu376 plays in modulating thioester polarization in the medium chain acyl-CoA dehydrogenase.     

Structural basis for the emission of violet bioluminescence from a W92F obelin mutant

Mutation of the Trp92 that is known to lie within the active site of the photoprotein obelin from Obelia longissima, results in a shift of the bioluminescence color from blue (lambda(max)=485 nm) to violet. The corrected spectrum shows a new band with lambda(max)=410 nm now contributing equally to the one at longer wavelength. The crystal structure of this W92F obelin determined at 1.72 A resolution shows that there is no significant change in the dimensions of the active site between WT obelin (recombinant Ca2+-regulated photoprotein from Obelia longissima) and the mutant. It is proposed that the bioluminescence spectral shift results from removal of a hydrogen bond from the indole of W92 nearby a hydroxyl belonging to the 6-phenyl substituent of the substrate coelenterazine. Propagation of this change through a conjugated bond system in the excited state of the product coelenteramide affects the coupling of the N1-position and the hydrogen-bonded Y138.     

Squid retinochrome

Retinochrome is a photosensitive pigment located primarily in the inner portions of the visual cells of cephalopods. Its absorption spectrum resembles that of rhodopsin, but its chromophore is all-trans retinal, which light isomerizes to 11-cis, the reverse of the situation in rhodopsin. The 11-cis photoproduct of retinochrome slowly reverts to retinochrome in the dark. The chromophoric site of retinochrome is more reactive than that of most visual pigments: (a) Hydroxylamine converts retinochrome in the dark to all-trans retinal oxime + retinochrome opsin. (by Sodium borohydride reduces it to N-retinyl opsin. (c) Lambda max of retinochrome shifts from 500 to 515 nm as the pH is raised from 6 to 10, with a loss of absorption above pH 8; meanwhile above this PH a second band appears at shorter wavelengths with lambda max 375 nm. These changes are reversible. (d) If retinochrome is incubated with all-trans 3-dehydroretinal (retinal2) in the dark, some 3-dehydroretinochrome (retinochrome2, lambda max about 515 nm) is formed. Conversely, when retinochrome2, made by adding all-trans retinal2 to bleached retinochrome or retinochrome opsin, is incubated in the dark with all-trans retinal some of it is converted to retinochrome. Retinal and 3-dehydroretinal therefore can replace each other as chromophores in the dark.     

Methane oxidation by cell-free extracts of Methylococcus capsulatus

The separation of two distinct forms of cytochrome P450 from adrenal cortex mitochondria has been achieved by the following steps; (1) lyophilisation (2) iso-octane extraction, (3) (NH(4))(2)SO(4) fractionation in the presence of sodium cholate. The fraction precipitating between 25-35 percent (NH(4))(2)SO(4) gave a difference spectrum with 11-deoxycorticosterone (11-DOC) but not with 20alpha-hydroxycholesterol (20alpha-HOC). This fraction showed high 11beta-hydroxylase activity but low activity for side chain cleavage of cholesterol (S.C.C.). The fraction precipitating between 45-60 percent (NH(4))(2)SO(4) gave a difference spectrum with 20alpha-HOC but not with 11-DOC and exhibited high S.C.C. activity but low 11beta-hydroxylase activity. The absorption spectrum of the 45-60 percent fraction indicated a preponderance of high spin hemoprotein (lambda(max) 395 nm).     

Modification of spectral sensitivities by screening pigments in the compound eyes of twilight-active fireflies (Coleoptera: Lampyridae)

1. ERG S(lambda) were determined in dark-adapted intact preparations of 6 North American firefly species (Photinus collustrans, marginellus, pyralis, macdermotti, scintillans and Bicellonycha wickershamorum) which restrict their flashing activity to twilight hours. The curves possess narrow (1/2 bandwidth = 50-60 nm) peaks in the yellow (560-580 nm) and a shoulder in the violet (370-420 nm), with a marked attenuation (1.4-2.2 log units) of sensitivity in the green (480-530 nm) region of the spectrum (Fig. 1). Two additional species (Photuris potomaca and frontalis) which initiate flashing at twilight and continue on late into the night (twi-night) possess broad sensitivity maxima around 560 nm (Fig. 3). 2. Selective adaptation experiments isolated near-UV and yellow in P. scintillans (Fig. 2). In the dorsal frontal region of the compound eyes in P. frontalis, high sensitivity existed only in the short wavelength region (near-UV and blue) with a maximum in the blue (lambda max 435 nm) (Fig. 4). 3. The in situ MSP absorption spectrum of the screening pigments was determined in preparations of firefly retina. a) Two kinds of dark brown granules were found in the clear zone region. These granules absorb all across the spectrum with a gradual increase in optical density in the shorter wavelength region in P. pyralis (Fig. 5). b) Besides dark granules, pink-to-red colored screening pigments were present in the vicinity of the rhabdoms. The absorption spectra of these pigments determined in five species were narrow (1/2 bandwidth = 50-80 nm) with species-specific differences in their peak absorption in the green at 525 nm, 510 nm, 512 nm and 517 nm in P. scintillans, macdermotti, collustrans and pyralis, respectively (Fig. 6). A similar pigment was found in P. marginellus with a lambda max at 512 nm (Fig. 7). In all cases, transmission increased both at long and short wavelengths, but more sharply in the long wavelength region (Figs. 6 and 7). Hence each twilight-restricted species has its own unique colored screening pigment. A yellow pigment whose absorption spectrum differed from those found in genus Photinus was found in twi-night active Photuris potomaca (lambda max 461 nm) and night-active P. versicolor (lambda max 456 nm). The transmission of the Photuris pigment increased sharply only in the long wave-length region (Fig. 8).(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of a strictly conserved active site tyrosine in cofactor genesis in the copper amine oxidase from Hansenula polymorpha

The copper amine oxidases catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain within the same active site. The contributions of individual active site amino acids to each of these chemical processes are being delineated. Previously, using the amine oxidase from the yeast Hansenula polymorpha (HPAO), mutations of a strictly conserved and structurally pivotal active site tyrosine (Y305) were studied and their effects on the catalytic cycle demonstrated [Hevel, J. M., Mills, S. A., and Klinman, J. P. (1999) Biochemistry 38, 3683-3693]. This study examines mutations at the same position for their effects on cofactor generation. While the Y305A mutation had moderate effects on the kinetics of catalysis (2.5- and 8-fold effects on k(cat) using ethylamine and benzylamine as substrates), the same mutation slows cofactor formation by approximately 45-fold relative to that of the wild-type (WT). Additionally, the Y305A mutant forms at least two species: primarily TPQ at lower pH and a species with a blue-shifted absorbance at high pH (lambda(max) = 400 nm). The 400 nm species does not react with phenylhydrazine or ethylamine and is stable toward pH buffer exchange, long-term storage (>3 weeks), incubation at high temperatures, or incubation with reductants and colorimetric peroxide quenching reagents. A similar species accumulates appreciably even at approximately neutral pH in the Y305F mutant, despite the fact that the rate of TPQ formation is reduced only 3-fold relative to that of WT HPAO. This small impact of Y305F on the rate of biogenesis contracts with a decrease in k(cat) (using ethylamine as the substrate) of 125-fold. The opposing effects of mutations at position 305 in biogenesis versus catalysis indicate that a single residue can be recruited for different roles during these processes.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.