Study of interaction of cytochrome P450 2B4 with riboflavin by fluorescence spectrometry

Fluorescence quenching of riboflavin by cytochrome P450 2B4 was used to probe the ligand--enzyme binding interaction ((lambda ex = 385 nm, lambda em = 520 nm). Riboflavin is a component of a flavoprotein NADPH dependent cytochrome P450 reductase, an essential electron carrier during cytochrome P450 catalysis. Fluorescence titration measurements revealed that cytochrome P450 2B4 and riboflavin formed a complex with an apparent Kd = 8.8 +/- 1 microM. The fluorescence intensity of riboflavin decreased upon the addition of cytochrome P450 2B4, which may be caused by the resonance excitation energy transfer from the fluorescent donor riboflavin to the cytochrome P450 2B4 heme acceptor. These data suggest that there may exist specific sites of binding of riboflavin with the protein globule of cytochrome P450 2B4.     

Membrane glycoproteins of Chlamydomonas eugametos flagella

The flagellar glycoproteins exposed on Chlamydomonas eugametos gametes were labeled by means of lactoperoxidase, diiodosulfanilic acid and chloramine T, and characterised in SDS-electrophoresis gels. The medium from gamete cultures contains particles (isoagglutinins) that agglutinate gametes of the opposite mating type. When crude preparations of these particles were subjected to isopycnic centrifugation in a caesium chloride gradient, two bands of particles were found. The lighter, active band consisted of membrane vesicles. The denser, inactive band consisted of cell wall material. The active band had the same glycoprotein composition as membrane vesicles artificially made from isolated flagella. Preparations of glagella were also separated on a caesium chloride cushion into pure flagella and cell wall material. The flagella, but not the cell wall material, isoagglutinated opposite gametes. Again the glycoprotein composition of pure flagella was similar to that of pure isoagglutinin vesicles. No difference was detected between the protein and glycoprotein compositions of flagella and isoagglutinins from both mating types.Abbreviations LPO lactoperoxidase - PB phosphate buffer - DISA diazotized ¹²⁵I-iodo-sulfanilic acid - SDS sodium dodecyl sulphate - CBD coomassie Brilliant Blue - PAS periodic acid Schiff     

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces changes in the heme spin state of microsomal cytochrome P-450

In vitro studies on the nature of interaction of the neurotoxin MPTP with hepatic microsomal cytochrome P-450 were carried out. Spectral perturbation studies showed nitrogenous ligand type binding between MPTP and cytochrome P-450 with a peak at 423 nm and a broad trough at 400 nm. Scatchard analysis of MPTP-cytochrome P-450 binding suggested that MPTP binds to at least 2 species of cytochrome P-450--a high affinity binding species with an apparent spectral dissociation constant (Ks) of 372 microM and a low affinity species with Ks of 37.6 mM. EPR studies confirmed that MPTP is a type II substrate for the forms of cytochrome P-450 with which it interacts and causes a shift from the high spin state of cytochrome P-450 to the low spin state. MPTP is, thus, likely to be an effective inhibitor of cytochrome P-450.     

Evolution of vitamin B2 biosynthesis: riboflavin synthase of Arabidopsis thaliana and its inhibition by riboflavin

A synthetic gene specifying the catalytic domain of the Arabidopsis thaliana riboflavin synthase was expressed with high efficiency in a recombinant Escherichia coli strain. The recombinant pseudomature protein was shown to convert 6,7-dimethyl-8-ribityllumazine into riboflavin at a rate of 0.027 s-1 at 25 degrees C. The protein sediments at a rate of 3.9 S. Sedimentation equilibrium analysis afforded a molecular mass of 67.5 kDa, indicating a homotrimeric structure, analogous to the riboflavin synthases of Eubacteria and fungi. The protein binds its product riboflavin with relatively high affinity (Kd =1.1 microM). Product inhibition results in a characteristic sigmoidal velocity versus substrate concentration relationship. Characterization of the enzyme/product complex by circular dichroism and UV absorbance spectroscopy revealed a shift of the absorption maxima of riboflavin from 370 and 445 to 399 and 465 nm, respectively. Complete or partial sequences for riboflavin synthase orthologs were analyzed from 11 plant species. In each case for which the complete plant gene sequence was available, the catalytic domain was preceded by a sequence of 1-72 amino acid residues believed to function as plastid targeting signals. Comparison of all available riboflavin synthase sequences indicates that hypothetical gene duplication conducive to the two-domain architecture occurred very early in evolution.     

Nuclear ribosomal RNA genes and algal phylogeny--the Chlamydomonas example

The nuclear ribosomal RNA genes (rDNA) of Chlamydomonas reinhardtii, C. moewusii and C. eugametos were examined with restriction endonuclease fragment and direct rRNA sequencing analyses. These comparative molecular data confirm similarity between C. moewusii and C. eugametos, and dissimilarity between the strains and C. reinhardtii. For C. moewusii and C. eugametos, the fragment analysis of digests with 16 (six base pair recognition site) restriction endonucleases revealed either no or minor differences. These minor differences appear to be confined to length and site variation in the rapidly evolving intergenic spacer region of the algal rDNA repeat unit. In contrast, patterns of digests for C. reinhardtii were completely different from those of C. moewusii and C. eugametos for all enzymes tested. Over two regions of the 18S ribosomal RNA (spanning approx. 300 bases) in C. moewusii and C. eugametos, we observed three possible base substitutions and no insertion/deletion events. The same comparison between C. reinhardtii and C. moewusii (or C. eugametos) revealed 31 base substitutions and eight insertion/deletion events. Overall, the rDNA comparisons support the proposed conspecificity of C. moewusii and C. eugametos, as well as the hypothesis that intraspecific variation in the algal ribosomal RNA coding region is minimal and that comparisons of rDNA sequences at higher taxonomic levels can be useful indicators of algal phylogeny. The degree of difference in the sequences of the 18S coding region between C. reinhardtii and C. moewusii or C. eugametos is comparable to that between an angiosperm and Equisetum and may reflect an ancient divergence between two species in one algal genus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent assay for riboflavin binding to cytochrome P450 2B4

The interactions between the hemoprotein cytochrome P450 2B4 (CYP 2B4) and riboflavin - a low molecular weight component of the flavoprotein NADPH-dependent cytochrome P450 reductase - were investigated by fluorescence spectroscopy. Riboflavin fluorescence quenching by cytochrome P450 2B4 was used to probe the ligand-enzyme binding (lambda(ex)=385 nm, lambda(em)=520 nm). Fluorescence titration experiments showed formation of a complex between cytochrome P450 2B4 and riboflavin with an apparent dissociation constant value, K(d)=8.8+/-1 microM. The fluorescence intensity of riboflavin was decreased with increasing the cytochrome P450 2B4 concentration, indicating the transfer of resonance excitation energy from riboflavin (energy donor) to the cytochrome P450 2B4 heme (energy acceptor). The data obtained are suggestive of the existence of riboflavin binding site(s) on the hemeprotein molecule.     

Inhibition of aromatase cytochrome P-450 by 10-oxirane and 10-thiirane substituted androgens. Implications for the structure of the active site

The mechanism of inhibition of estrogen synthetase (P-450arom) by 19R- and 19S-isomers of 10-oxiranyl-and 10-thiiranyl-4-estrene-3,17-dione was investigated using human placental microsomes and purified enzyme preparations. The 19R-isomers were potent inhibitors and exhibited affinities 36-fold (10-oxirane) and 80-fold (10-thiirane) greater than the respective 19S-isomers. Kinetic experiments showed that inhibition by the 19R-isomers is competitive with respect to substrate; inhibition constants for the (19R)-10-oxirane (Ki = 10 nM) and the 19R-10-thiirane (Ki = 2 nM) indicate that each binds with greater affinity than the androgen substrates androstenedione and testosterone. Inhibition time courses and kinetic data were consistent with high affinity, reversible binding. Spectral titrations of microsomal preparations and purified P-450arom showed that binding of the 19R-isomers shifts the Soret maximum of the ferric enzyme to 411 nm (10-oxirane) or 425 nm (10-thiirane); addition of excess androstenedione reversed the spectral changes, producing the high spin form of the enzyme with a Soret peak at 393 nm. These spectral shifts suggest that the oxygen atom of the 10-oxirane and the sulfur atom of the 10-thiirane are bound to the heme iron in the inhibitor complexes. These results suggest that the high affinities of the inhibitors arise from their dual interaction with the androgen binding site and with the heme. Coordination of the C19 heteroatom to the heme indicates that C19 of androgen substrates may be positioned sufficiently close to the heme to allow direct attack by an iron-bound oxidant. Stereoselective binding of the 19R-isomers by P-450arom further suggests that the heme is likely to be positioned above C1 and C2 of the A ring.     

Flavin affinity chromatography: General methods for purification of proteins that bind riboflavin

Analogs of riboflavin that were altered at positions N(3), 8α, and N(10) of the 7,8-dimethylisoalloxazine ring were immobilized by covalent attachment to aminoalkylated agarose and polyacrylamide beads. These materials were used for affinity chromatographic purification of the riboflavin-carrier protein from egg white, egg yolk, and blood from laying hens, of flavokinase from rat liver, and of partially purified flavodoxin from Azotobacter vinelandii (FMN). The apo-carrier protein, which tightly complexes riboflavin (K_d ≈ 2 nm), was bound by the N(3)-, 8α-, and N(10)-flavinyl beads and was selectively displaced in moderate to high yield by 10 μm riboflavin or 1 m NaCl at pH 3.5. Flavokinase, which complexes less tightly with riboflavin (K_m ≈ 12 μm), was bound by the 8α- and N(10)-flavinyl beads. Binding to the latter was sufficiently tight that the addition of riboflavin was needed to displace flavokinase from the beads. The A. vinelandii flavodoxin, which normally complexes riboflavin 5′-phosphate (K₃ ≈ 5 nm) but less avidly complexes riboflavin (K_d ≈ 0.6 μm), was bound by the N(10)-flavinyl beads and eluted in low yield upon addition of FMN; most of the apoprotein denatured on the column despite the inclusion of thiol-protecting reagents. These flavin affinity materials may be generally useful for isolating a variety of other proteins that bind riboflavin.     

Two group I introns with long internal open reading frames in the chloroplast psbA gene of Chlamydomonas moewusii.

We report the nucleotide sequence of the chloroplast psbA gene encoding the 32 kilodalton protein of photosystem II from Chlamydomonas moewusii. Like its land plant homologues, this green algal protein consists of 353 amino acids. The C. moewusii psbA gene is composed of three exons containing 252, 11 and 90 codons and of two group I introns containing 2363 and 1807 nucleotides. Each of the introns features an internal open reading frame (ORF) that potentially encodes a basic protein of more than 300 residues. The primary sequences of the putative intron-encoded proteins are unrelated and none of them shares conserved elements with any of the proteins predicted from the group I intron sequences published so far. The first C. moewusii intron is inserted at the same position as the fourth intron of the psbA gene from Chlamydomonas reinhardtii; the second intron lies at a novel site downstream of this position. On the basis of their RNA secondary structures, the C. moewusii introns 1 and 2 can be assigned to subgroups IA and IB, respectively. However, intron 1 is not typical of subgroup IA introns, its most unusual feature being the location of the ORF in the "loop L5" region. To our knowledge, this is the first time that an ORF is located in this region of the group I intron structure.     

Characterisation and cloning of a calmodulin-like domain protein kinase from Chlamydomonas moewusii (Gerloff )

Calcium-stimulated protein kinase activity in the flagella of the green alga Chlamydomonas moewusii (Gerloff) was characterised. Using SDS-PAGE and an on-blot phosphorylation assay, a 65-kDa protein was identified as the major calcium-stimulated protein kinase. Its activity was directly stimulated by calcium, a characteristic of the calmodulin-like domain protein kinases (CDPKs). Monoclonal antibodies raised against the CDPKα from soybean cross-reacted with the 65-kDa protein in the flagella, and also with other proteins in the flagellum and cell body. The same monoclonal antibodies were used to screen a C. moewusii cDNA expression library in order to isolate CDPK cDNAs from C. moewusii. The CCK1 cDNA encodes a protein with a kinase and calmodulin-like domain linked by a junction domain typical of CDPKs. From Southern analyses, evidence was obtained for a CDPK gene family in C. moewusii and C. reinhardtii. Received: 9 July 1996 / Accepted: 13 November 1996     

Biosynthesis of riboflavin: 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe.

A cDNA sequence from Schizosaccharomyces pombe with similarity to 6,7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia coli strain. The recombinant protein is a homopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibrium centrifugation (it sediments at an apparent velocity of 5.0 S at 20 degrees C). The protein has been crystallized in space group C2221. The crystals diffract to a resolution of 2.4 A. The enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy- 2-butanone 4-phosphate. Steady-state kinetic analysis afforded a vmax value of 13 000 nmol.mg-1.h-1 and Km values of 5 and 67 microm for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, respectively. The enzyme binds riboflavin with a Kd of 1.2 microm. The fluorescence quantum yield of enzyme-bound riboflavin is < 2% as compared with that of free riboflavin. The protein/riboflavin complex displays an optical transition centered around 530 nm as shown by absorbance and CD spectrometry which may indicate a charge transfer complex. Replacement of tryptophan 27 by tyrosine or phenylalanine had only minor effects on the kinetic properties, but complexes of the mutant proteins did not show the anomalous long wavelength absorbance of the wild-type protein. The replacement of tryptophan 27 by aliphatic amino acids substantially reduced the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.     

Reconstitution of biological activity in isoagglutinins fromChlamydomonas eugametos

Gametes ofChlamydomonas eugametos produce membrane vesicles, called isoagglutinins, which are shed into the culture fluid. It is assumed that they originate from the flagellar membrane for, like flagella, they can bind to the flagellar surface of gametes of the opposite mating type (mt). The composition ofmt ^- isoagglutinin was investigated with respect to this agglutinability. When the agglutination factor present on the surface ofmt ^- isoagglutinins (PAS-1.2) was removed, together with other membrane bound glycoproteins, the membrane vesicles were rendered inactive. They could be reactivated however by incubation with the extracted glycoproteins in a time-and concentration-dependent manner. The agglutination factor proved to be necessary yet sufficient in itself for the reactivation process to occur. Experiments with CsCl density gradients showed that the agglutination factor truly bound to the vesicles during reactivation. Inactivated vesicles derived frommt ⁺ gametes could be reactivated to gainmt ^- properties. Reactivation was inhibited by prior treatment with trypsin. The results indicate that the agglutination factor inmt ^- isoagglutinins is an extrinsic membrane protein bound to an intrinsic proteinaceous receptor.Abbreviations GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PAS periodic acid Schiff     

Flavin binding to the high affinity riboflavin transporter RibU

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B(2)) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655-5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (K(d) for riboflavin is 0.6 nM), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure.     

Extensive gene rearrangements in the chloroplast DNAs of Chlamydomonas species featuring multiple dispersed repeats

We have constructed a physical and gene map for the chloroplast DNA (cpDNA) of the unicellular green alga Chlamydomonas gelatinosa, a close relative of Chlamydomonas reinhardtii. At 285 kb, the C. gelatinosa cpDNA is 89 kb larger than its C. reinhardtii counterpart. The alterations in the order of 77 genes on the cpDNAs of these green algae are attributable to nine inversions and one event of expansion/contraction of the inverted repeat. These rearrangements are much more extensive than those previously reported between the cpDNAs of the closely related Chlamydomonas moewusii and Chlamydomonas pitschmannii. Because the divergence level of the C. gelatinosa and C. reinhardtii chloroplast-encoded large subunit rRNA gene sequences is equivalent to that of the corresponding C. moewusii and C. pitschmannii sequences, our results may suggest that, in the same period of time, there have been more numerous rearrangements in the lineage comprising C. gelatinosa and C. reinhardtii than in the lineage comprising C. moewusii and C. pitschmannii. Alternatively, given that substitution rates in chloroplast genes are not necessarily uniform across lineages, the extensive rearrangements between the C. gelatinosa and C. reinhardtii cpDNAs may reflect a longer divergence period for this pair of Chlamydomonas species compared to that for the C. moewusii/C. pitschmannii pair. We have also found that, like its C. reinhardtii homologue but unlike its C. moewusii and C. pitschmannii counterparts, the C. gelatinosa cpDNA features a large number of dispersed repeated sequences that are readily detectable by Southern blot hybridization with homologous fragment probes. Assuming that the two pairs of closely related Chlamydomonas species diverged at about the same time, these data suggest that the susceptibility of Chlamydomonas cpDNAs to rearrangements is correlated with the abundance of repeated sequences. Preliminary characterization of a 345-bp C. gelatinosa cpDNA region containing a repeated sequence by both DNA sequencing and Southern blot analysis has revealed no sequence homology between this region and the cpDNAs of C. reinhardtii and other Chlamydomonas species.      

ELECTRON AND PHASE-CONTRAST MICROSCOPY OF SEXUAL REPRODUCTION IN CHLAMYDOMONAS MOEWUSII1

The sexual process of C. moewusii from gametic activation through germination of the zygote has been studied with phase-contrast and electron microscopy. Long strands emerging from the gametic flagellar tips are the site of early flagellar attraction which is followed by union of compatible flagella within common flagellar sheaths. The gametic connecting strand is formed by coordinated elongation of the plasma papillae of a gametic pair and the penetration of the former through their wall papillae while the flagella are in intimate association. After the free-swimming period, the gametic pairs aggregate in a second period of clumping. The connecting strand is abscised and extruded during plas-mogamy as are the flagellar basal bodies. Evidence is presented which suggests union of the gametic plastids, and stages in karyogamy are illustrated. Formation of the wall layers, accumulation of starch and lipids, and changes in plastid organization in the maturing and germinating zygote are described as is the formation of the gonal walls.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.     

ON SEXUAL AGGLUTINATION AND MATING-TYPE SUBSTANCES IN ISOGAMOUS DIOECIOUS CHLAMYDOMONADS. III. THE SENSITIVITY OF SEX CELL CONTACT TO VARIOUS ENZYMES

The components responsible for sex cell contact in dioecious Chlamydomonas species have been characterized by their sensitivity to enzymes. The mating-type substances, which are glycoprotein complexes according to the analysis of the corresponding isoagglutinins, were incubated with proteases, glycosidases, and with sulfatase. An enzyme may or may not affect both gamete types of a species or it may discriminate between the sexes, eliminating one mating-type activity only. In no case was an incapacitation of a given gamete type based upon the detachment of the proper contact component as an isoagglutinin or as a functionally univalent structure with a specific receptor-blocking power against the complementary type. Inactivating enzymes seem to interfere with the surface configuration essential for gamete contact or to eliminate actual contact sites. The compilation of data for six taxa reveals a complex sensitivity pattern of the gametes’ agglutinability. Two types of interactions appear to occur. In C. reinhardti and C. chlamydogama the contact mechanism seems to depend on an interaction between two proteinaceous structures. In C. eugametos and C. moewusii definite proof was obtained for a contact based upon a carbohydrate-protein interaction. In the (+) sexes of C. eugametos and C. moewusii syngen II, α-glycosidically bound terminal mannose residues are essential for the contact. Neither β-galactosidase, neuraminidase, or sulfatase incapacitate the mating-type substances in situ.     

Photodegradation of riboflavin in neonates

The biologically most important flavins are riboflavin and its related nucleotides, all highly sensitive to light. It is because of its photoreactivity and its presence in almost all body fluids and tissues that riboflavin assumes importance in phototherapy of neonatal jaundice. The absorption maxima of both bilirubin and riboflavin in the body are nearly identical: 445-450 (447) nm. In consequence, blue visible light will cause photoisomerization of bilirubin accompanied by photodegradation of riboflavin. This results in diminished erythrocyte glutathione reductase, which indicates generalized tissue riboflavin deficiency and red cell lysis. Single- and double-strand breaks in intracellular DNA have occurred with phototherapy. This light exposure of neonates may result also in alterations of bilirubin-albumin binding in the presence of both riboflavin and theophylline (the latter frequently given to prevent neonatal apnea). Many newborns, especially if premature, have low stores of riboflavin at birth. The absorptive capacity of premature infants for enteral riboflavin is likewise reduced. Consequently, inherently low stores and low intake of riboflavin plus phototherapy for neonatal jaundice will cause a deficiency of riboflavin at a critical period for the newborn. Supplementation to those infants most likely to develop riboflavin deficiency is useful, but dosage, time, and mode of administration to infants undergoing phototherapy must be carefully adjusted to avoid unwanted side effects.     

The solution structure of the N-terminal domain of riboflavin synthase

The structure of the amino-terminal domain of Escherichia coli riboflavin synthase (RiSy) has been determined by NMR spectroscopy with riboflavin as a bound ligand. RiSy is functional as a 75 kDa homotrimer, each subunit of which consists of two domains which share very similar sequences and structures. The N-terminal domain (RiSy-N; 97 residues) forms a 20 kDa homodimer in solution which binds riboflavin with high affinity. The structure features a six-stranded antiparallel beta-barrel with a Greek-key fold, both ends of which are closed by an alpha-helix. One riboflavin molecule is bound per monomer in a site at one end of the barrel which is comprised of elements of both monomers. The structure and ligand binding are similar to that of the FAD binding domains of ferrodoxin reductase family proteins. The structure provides insights into the structure of the whole enzyme, the organisation of the functional trimer and the mechanism of riboflavin synthesis. C48 from the N-terminal domain is identified as the free cysteine implicated in a nucleophilic role in the synthesis mechanism, while H102 from the C-terminal domains is also likely to play a key role. Both are invariant in all known riboflavin synthase sequences.     

Effect of riboflavie and cyanide on blue light induction of betacyanin formation in Amamnthus caudatus seedlings

The role of phyto chrome and flavins in blue light induction of betacyanin formation was studied in etiolated, three-day-old Amaranthus caudatus L. seedlings, using the criterion of far-red reversibility and exogenously applied riboflavin and KCN. The effect of riboflavin was studied using high fluence rate blue light (42.7 :nmol m⁻²s⁻¹nm⁻¹ at 450 nm). When present in the incubation medium during illumination, riboflavin promoted the far-red reversibility with short light treatments and suppressed the inductive action of continuous illumiaation. If added after light treatments, it promoted betacyanin formation. The filtration of blue light through the riboflavin solution caused profound changes in light quality without affecting the far-red reversibility after 30 mm illumination. The effect of 1 mM KCN was tested with 70'% lower fluence of blue light. Cyanide caused the suppression of the inductive effect with 5 min blue light, which was accompanied by an enhancement of betacyanin induction by the terminal far-red light pulse. With 30 min blue light, however, it caused the appearance of far-red reversibility. The inductive effect of continuous blue illumination was slightly promoted by this Inhibitor. These results demonstrate that the effect of blue light on the pbyto chrome system is complex, whereas the physiological (inductive) action of the flavin triplet state is limited to low fluence, short blue light treatments.