Tryptophan 512 is sensitive to conformational changes in the rigid relay loop of smooth muscle myosin during the MgATPase cycle

To examine the structural basis of the intrinsic fluorescence changes that occur during the MgATPase cycle of myosin, we generated three mutants of smooth muscle myosin motor domain essential light chain (MDE) containing a single conserved tryptophan residue located at Trp-441 (W441-MDE), Trp-512 (W512-MDE), or Trp-597 (W597-MDE). Although W441- and W597-MDE were insensitive to nucleotide binding, the fluorescence intensity of W512-MDE increased in the presence of MgADP-berellium fluoride (BeF(X)) (31%), MgADP-AlF(4)(-) (31%), MgATP (36%), and MgADP (30%) compared with the nucleotide-free environment (rigor), which was similar to the results of wild type-MDE. Thus, Trp-512 may be the sole ATP-sensitive tryptophan residue in myosin. In addition, acrylamide quenching indicated that Trp-512 was more protected from solvent in the presence of MgATP or MgADP-AlF(4)(-) than in the presence of MgADP-BeF(X), MgADP, or in rigor. Furthermore, the degree of energy transfer from Trp-512 to 2'(3')-O-(N-methylanthraniloyl)-labeled nucleotides was greater in the presence of MgADP-BeF(X), MgATP, or MgADP-AlF(4)(-) than MgADP. We conclude that the conformation of the rigid relay loop containing Trp-512 is altered upon MgATP hydrolysis and during the transition from weak to strong actin binding, establishing a communication pathway from the active site to the actin-binding and converter/lever arm regions of myosin during muscle contraction.     

Actin-induced closure of the actin-binding cleft of smooth muscle myosin

The putative actin-binding interface of myosin is separated by a large cleft that extends into the base of the nucleotide binding pocket, suggesting that it may be important for mediating the nucleotide-dependent changes in the affinity for myosin on actin. We have genetically engineered a truncated version of smooth muscle myosin containing the motor domain and the essential light chain-binding region (MDE), with a single tryptophan residue at position 425 (F425W-MDE) in the actin-binding cleft. Steady-state fluorescence of F425W-MDE demonstrates that Trp-425 is in a more solvent-exposed conformation in the presence of MgATP than in the presence of MgADP or absence of nucleotide, consistent with closure of the actin-binding cleft in the strongly bound states of MgATPase cycle for myosin. Transient kinetic experiments demonstrate a direct correlation between the rates of strong actin binding and the conformation of Trp-425 in the actin-binding cleft, and suggest the existence of a novel conformation of myosin not previously seen in solution or by x-ray crystallography. Thus, these results directly demonstrate that: 1) the conformation of the actin-binding cleft mediates the affinity of myosin for actin in a nucleotide-dependent manner, and 2) actin induces conformational changes in myosin required to generate force and motion during muscle contraction.     

Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.     

Nucleotide dependent intrinsic fluorescence changes of W29 and W36 in smooth muscle myosin

The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin. In this study, an MDE containing all the endogenous tryptophans except W512 (W512 KO-MDE) decreases in intrinsic fluorescence upon nucleotide binding, demonstrating that the intrinsic fluorescence enhancement of smooth muscle myosin is not solely due to W512. Candidates for the observed quench of intrinsic fluorescence in W512 KO-MDE include W29 and W36. Whereas the intrinsic fluorescence of W36-MDE is only slightly sensitive to nucleotide binding, that of W29-MDE is paradoxically both quenched and blue-shifted upon nucleotide binding. Steady-state and time-resolved experiments suggest that fluorescence intensity changes of W29 involve both excited-state and ground-state quenching mechanisms. These results have important implications for the role of the N-terminal domain (residues 1-76) in smooth muscle myosin in the molecular mechanism of muscle contraction.     

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.     

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.     

Kinetic characterization of the function of myosin loop 4 in the actin-myosin interaction

Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (DeltaAL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.     

Kinetic mechanism and regulation of myosin VI.

Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of P(i) release, which lowers the K(ATPase) but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins.     

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.     

Mechanoenzymatic characterization of human myosin Vb

There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V(max) and K(actin) of the actin-activated ATPase activity of HuM5B were 9.7 +/- 0.4 s(-)(1) and 8.5 +/- 0.1 microM, respectively. K(actin) and K(ATP) of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 +/- 0.5 s(-)(1), which was comparable to the V(max) of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 +/- 0.03 microm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.     

Exciton coupling effects and conformational change of perhexyloligosilanes with optically active methyl(1-naphthyl)phenylsilyl terminals

Perhexyloligosilanes (R,R)-(+)-MeNpPhSi*(Hex(2)Si)(n)Si*PhNpMe (n = 2; (R,R)-(+)-4a, n = 4; (R,R)-(+)-6a, n = 6; (R,R)-(+)-8a) with chiral methyl(1-naphthyl)phenylsilyl terminals were synthesized and characterized. The absorption wavelengths lambda(max) by (1)L(a,Ph) transition of phenyl chromophore conjugated with oligosilane units in (R,R)-(+)-4a - (R,R)-(+)-8a show bathochromic shift of about 3-4 nm compared with those of the alpha,omega-phenyl substituted perhexyloligosilanes Ph(Hex(2)Si)(m)Ph (m = 4; 4b, m = 6; 6b, m = 8; 8b) having the same silicon chain length. Longer chain length induces the separated lambda(max) of (1)L(a,Ph) from (1)B(b,Np) of naphthyl chromophore with positive exciton chiralities. In (R,R)-(+)-8a, although the extremum wavelengths lambda(ext) of exciton coupling between (1)B(b,Np) and (1)L(a,Ph) are separated by about 80 nm, the compound retains the positive exciton chirality, which provides definite information on the absolute configuration of terminal chiral silicon atoms. Bulky terminal substituents and lowering the temperature affect the conformation of the main chain, inducing extended silicon backbone structure.     

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).     

Isolation and characterization of mutants of firefly luciferase which produce different colors of light

The luciferase cDNA from the 'Genji' firefly, Luciola cruciata, was mutated with hydroxylamine to isolate mutant luciferases. Some of the isolated mutant enzymes produced different colors of light, ranging from green to red. Five such mutants, producing green (lambda max = 558 nm), yellow-orange (lambda max = 595 nm), orange (lambda max = 607 nm) and red light (lambda max = 609 and 612 nm), were analyzed. The mutations were found to be single amino acid changes, from Val239 to Ile, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser and His433 to Tyr respectively.     

Amino acid residues critical for the specificity for betaine aldehyde of the plant ALDH10 isoenzyme involved in the synthesis of glycine betaine

Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant K(m)(BAL) increases and V(max)/K(m)(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the V(max)/K(m)(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.     

Reaction of Pseudomonas fluorescens kynureninase with beta-benzoyl-L-alanine: detection of a new reaction intermediate and a change in rate-determining step

Beta-benzoyl-DL-alanine was synthesized from alpha-bromoacetophenone and diethyl acetamidomalonate. The racemic amino acid was resolved by carboxypeptidase A-catalyzed hydrolysis of the N-trifluoroacetyl derivative. Beta-benzoyl-L-alanine is a good substrate of kynureninase from Pseudomonas fluorescens, with k(cat) and k(cat)/K(m) values of 0.7 s(-1) and 8.0 x 10(4) M(-1) s(-1), respectively, compared to k(cat) = 16.0 s(-1) and k(cat)/K(m) = 6.0 x 10(5) M(-1) s(-1) for L-kynurenine. In contrast to the reaction of L-kynurenine, beta-benzoyl-L-alanine does not exhibit a significant solvent isotope effect on k(cat) ((H)k/(D)k = 0.96 +/- 0.06). The pre-steady-state kinetics of the reaction of beta-benzoyl-L-alanine were investigated by rapid scanning stopped-flow spectrophotometry. The spectra show the formation of a quinonoid intermediate, with lambda(max) = 490 nm, in the dead time of the instrument, which then decays, with k = 210 s(-1), to form a transient intermediate with lambda(max) at 348 nm. In the presence of benzaldehyde, the 348 nm intermediate decays, with k = 0.7 s(-1), to form a quasistable quinonoid species with lambda(max) = 492 nm. Previous studies demonstrated that benzaldehyde can trap an enamine intermediate formed after the C(beta)-C(gamma) bond cleavage [Phillips, R. S., Sundararaju, B., and Koushik, S. V. (1998) Biochemistry 37, 8783-8789]. Thus, the 348 nm intermediate is kinetically competent. The position of the absorption maximum and shape of the band is consistent with a PMP-ketimine intermediate. The results from chemical quenching analysis do not show a burst of benzoate and, thus, also support the formation of benzoate as the rate-determining step. These data suggest that, in contrast to L-kynurenine, for which the rate-determining step was shown to be deprotonation of the pyruvate-ketimine intermediate [Koushik, S. V., Moore, J. A., III, Sundararaju, B., and Phillips, R. S. (1998) Biochemistry 37, 1376-1382], the rate-determining step in the reaction of beta-benzoyl-L-alanine with kynureninase is C(beta)-C(gamma) bond cleavage.     

Synthesis, structure and spectroscopic properties of two models for the active site of the oxygenated state of cytochrome P450 [corrected

Two dioxygen adducts of thiolato-iron(II) porphyrins, [K(222)][Fe(TPpivP)(SC6HF4)(O2)] 1a and [Na(18c.6)][Fe(TPpivP)(SC6HF4)(O2)] 2 were synthesized by reaction of O2 with five-coordinate, high-spin, cryptated alkali metal thiolato-iron(II) 'picket fence' porphyrinate. They were characterized by visible and infrared spectroscopy: lambda max (log epsilon) = 360 nm (4), 427 nm (4.69), 560 nm (3.69), 610 nm (3.40) for both compounds; v(16O-16O) = 1139 cm-1 in chlorobenzene and fluorobenzene for 1a and 2. Single crystals of composition [K(222)][Fe(TPpivP)(SC6HF4)(O2)].[K(222)](SC6HF4)(C 6H5Cl)(H2O) 1b were obtained by diffusion of pentane/xylene mixtures into chlorobenzene solutions of 1a at -5 degrees C. Single crystals of composition [Na(18c.6)][Fe(TPpivP)(SC6HF4)(O2)] were obtained by slow diffusion of pentane into benzene solutions of 2. Structures of 1b and 2 were studied at 20 degrees C (1b) and -100 degrees C (1b and 2). 1b: space group P2(1)/c (monoclinic), a = 16.806(5) A (1.6806 nm), b = 14.331(4) A (1.4331 nm), c = 52.000(15) A (5.2000 nm), beta = 92.95(2) degrees, V = 12.507 A3 (12.507 nm3), Z = 4, Dcal = 1.28 g.cm-3 (t = 20 degrees C). The final R1 factor was 0.085 for 5238 reflections having I greater than 3 sigma(I). 2: space group P2(1)/c (monoclinic), a = 13.107(3) A (1.3107 nm), b = 27.055(4) A (2.7055 nm), c = 25.029(4) A (2.5029 nm), beta = 96.84(2) degrees, V = 8812 A3 (8.812 nm3), Z = 4, Dcal = 1.18 g.cm-3 (t = -100 degrees C). The final R1 factor was 0.088 for 6587 reflections having I greater than 3 sigma(I). The iron atom is, in both compounds, bonded to the four porphyrinato nitrogens (Np), the sulfur atom of the axial thiolate and one oxygen atom of the axially end-on bonded dioxygen molecule. The average Fe-Np distance found in 1b [1.994(4) A, 0.1994 nm] is not significantly different from that found in 2 [1.993(3) A, 0.1993 nm]. The Fe-S bond length is 2.367(3) A (0.2367 nm) in 1b and 2.365(2) A (0.2365 nm) in 2. The Fe-O1 distances with the oxygen atom of O2 bonded to iron are respectively 1.837(9) A (0.1837 nm) and 1.850(4) A (0.1850 nm). The end-on bonded O2 molecule is disordered in both complexes 1b and 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

O2- and alpha-ketoglutarate-dependent tyrosyl radical formation in TauD,an alpha-keto acid-dependent non-heme iron dioxygenase

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.     

Mycosporine-like amino acid (MAAs) production by Heterocapasa sp. (Dinophyceae) in indoor cultures

The possibility of using mycosporine-like amino acids (MAAs), with an apparent sunscreen function in nature, as ultraviolet radiation (UVR) blockers to prevent skin injury has been raised by diverse authors. Production of MAAs by the dinoflagellate Heterocapsa sp. (Dinophyceae) is shown here. Three major peaks with absorption maxima at 330.8, 332.0 and 333.2 nm were detected by high performance liquid chromatography (HPLC) analysis of methanolic extracts in all tested conditions. Analysis of crude extract by mass spectroscopy with electrospray ionization (MS-EI) showed a set of molecular ions ([M+H](+)) with main peaks being at m/z 242.4, 288.4, 303.3 and 333.3 u.m.a. According to these data, along with retention times, the MAA profile of Heterocapsa sp. is assumed to be composed of shinorine (lambda(max)=334 nm), mycosporine-2-glycine (lambda(max)=331 nm) and palythinol (lambda(max)=332 nm). A constitutive MAA content of about 4 microg (10(6) cells)(-1) was measured under exposure to PAR only. A maximal accumulation of MAA per culture volume of 1.1 mg l(-1) was obtained after 72 h of exposure to PAR+UVA, while the highest production rate (0.025 mg l(-1) h(-1)) was computed after 24 h of exposure to PAR+UVA+UVB.     

Kinetic resolution of a tryptophan-radical intermediate in the reaction cycle of Paracoccus denitrificans cytochrome c oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.     

Eye function of Mysidacea (Crustacea) in the northern Baltic Sea

Eye spectral sensitivity, [S(lambda)], was measured in seven northern Baltic mysid species using an electroretinogram technique. Their S(lambda) curves were compared with the spectral distribution of underwater light at their normal habitats. In the littoral species Neomysis integer, Praunus flexuosus and Praunus inermis, the S(lambda) maxima, [S(lambda)(max)], were in the wavelength-bands of 525-535, 505-515 and 520-530 nm respectively. The neoimmigrant species Hemimysis anomala had a S(lambda)(max) around 500 nm and high sensitivity at 393 nm, possibly indicating UV-sensitivity. S(lambda) of the pelagic species Mysis mixta and Mysis relicta sp. II was at about 505-520 nm. M. relicta sp. I from Pojoviken Bay and fresh water humic Lake P??j?rvi had S(lambda)(max) at approximately 550 nm and 570 nm respectively. This is in accordance with a similar long-wavelength shift in light transmittance of the respective waters. The eyes of the latter population were also damaged by strong light. The pontocaspian neoimmigrant H. anomala is clearly adapted to waters transmitting more blue light.