Flavin Mononucleotide Binding Research Articles

Iron-Sulfur Cluster N5 Is Coordinated by an HXXXCXXCXXXXXC Motif in the NuoG Subunit of Escherichia coli NADH:Quinone Oxidoreductase (Complex I)

NADH:quinone oxidoreductase (complex I) plays a central role in cellular energy metabolism, and its dysfunction is found in numerous human mitochondrial diseases. Although the understanding of its structure and function has been limited, the x-ray crystal structure of the hydrophilic part of Thermus thermophilus complex I recently became available. It revealed the localization of all redox centers, including 9 iron-sulfur clusters and their coordinating ligands, and confirmed the predictions mostly made by Ohnishi et al. (Ohnishi, T., and Nakamaru-Ogiso, E. (2008) Biochim. Biophys. Acta 1777, 703-710) based on various EPR studies. Recently, Yakovlev et al. (Yakovlev, G., Reda, T., and Hirst, J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12720-12725) claimed that the EPR signals from clusters N4, N5, and N6b were misassigned. Here we identified and characterized cluster N5 in the Escherichia coli complex I whose EPR signals had never been detected by any group. Using homologous recombination, we constructed mutant strains of H101A, H101C, H101A/C114A, and cluster N5 knock-out. Although mutant NuoEFG subcomplexes were dissociated from complex I, we successfully recovered these mutant NuoCDEFG subcomplexes by expressing the His-tagged NuoCD subunit, which had a high affinity to NuoG. The W221A mutant was used as a control subcomplex carrying wild-type clusters. By lowering temperatures to around 3 K, we finally succeeded in detecting cluster N5 signals in the control for the first time. However, no cluster N5 signals were found in any of the N5 mutants, whereas EPR signals from all other clusters were detected. These data confirmed that, contrary to the misassignment claim, cluster N5 has a unique coordination with His(Cys)(3) ligands in NuoG.

Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus.

Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 66.49, c = 121.32 A. X-ray data sets have been collected to 2.17 A resolution.

Methionine synthase reductase undergoes phosphate‐sensitive loss of flavin mononucleotide upon dilution in vitro

Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one‐carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Methionine synthase reductase (MSR) is absolutely required for methionine synthase activity in vivo. MSR is a dual flavoprotein, containing one mol of FAD and one mol of FMN per mol of enzyme. While free FMN is highly fluorescent, enzyme‐bound FMN fluorescence is quenched. Flavin mononucleotide (FMN) binding in MSR is weak compared to other dual flavin‐containing enzymes (Kd = ~85 μM; 0 mM Pi), especially in the presence of phosphate (Kd = ~450 μM; 50 mM Pi) at constant ionic strength. Release of FMN from MSR is associated with loss of enzyme activity, as determined by cytochrome c reductase assay, while FMN‐reconstituted enzyme regains all activity. We found that the presence of 50 mM phosphate increased the overall loss of reductase activity upon dilution by ~50% and decreased the rate of rebinding of FMN ~5‐fold compared to phosphate free buffer. These findings point to a mechanism by which MSR may be inactivated under conditions that limit the availability of FMN in vivo in a phosphate sensitive manner.

A Novel Chromate Reductase from Thermus scotoductus SA-01 Related to Old Yellow Enzyme

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.

In Vivo Phosphorylation Site Mapping and Functional Characterization of Arabidopsis Phototropin 1

Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser(58), Ser(85), Ser(350), and Ser(410)), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a -NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that S-nitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975/990, and 1047/1049 as being susceptible to S-nitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse.

AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles.

A blue light (BL) receptor was discovered in stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae). Two homologs were identified in Vaucheria; each has one basic region/leucine zipper (bZIP) domain and one light-oxygen-voltage (LOV)-sensing domain. We named these chromoproteins AUREOCHROMEs (AUREO1 and AUREO2). AUREO1 binds flavin mononucleotide via its LOV domain and forms a 390-nm-absorbing form, indicative of formation of a cysteinyl adduct to the C(4a) carbon of the flavin mononucleotide upon BL irradiation. The adduct decays to the ground state in approximately 5 min. Its bZIP domain binds the target sequence TGACGT. The AUREO1 target binding was strongly enhanced by BL treatment, implying that AUREO1 functions as a BL-regulated transcription factor. The function of AUREO1 as photoreceptor for BL-induced branching is elucidated through RNAi experiments. RNAi of AUREO2 unexpectedly induces sex organ primordia instead of branches, implicating AUREO2 as a subswitch to initiate development of a branch, but not a sex organ. AUREO sequences are also found in the genome of the marine diatom Thalassiosira pseudonana (Bacillariophyceae), but are not present in green plants. AUREOCHROME therefore represents a BL receptor in photosynthetic stramenopiles.

Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH:ubiquinone oxidoreductase (complex I)

Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with K d (∼10 −8 M at pH 10.0).

N- and C-Terminal Flanking Regions Modulate Light-Induced Signal Transduction in the LOV2 Domain of the Blue Light Sensor Phototropin 1 from Avena sativa,

Light sensing by photoreceptors controls phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Understanding the molecular mechanism by which these processes are regulated requires a quantitative description of photoreceptor dynamics. We focus on a light-driven signal transduction mechanism in the LOV2 domain (LOV, light, oxygen, voltage) of the blue light photoreceptor phototropin 1 from Avena sativa (oat). High-resolution crystal structures of the dark and light states of an oat LOV2 construct including residues Leu404 through Leu546 (LOV2 (404-546)) have been determined at 105 and 293 K. In all four structures, LOV2 (404-546) exhibits the typical Per-ARNT-Sim (PAS) fold, flanked by an additional conserved N-terminal turn-helix-turn motif and a C-terminal flanking region containing an amphipathic Jalpha helix. These regions dock on the LOV2 core domain and bury several hydrophobic residues of the central beta-sheet of the core domain that would otherwise be exposed to solvent. Light structures of LOV2 (404-546) reveal that formation of the covalent bond between Cys450 and the C4a atom of the flavin mononucleotide (FMN) results in local rearrangement of the hydrogen-bonding network in the FMN binding pocket. These rearrangements are associated with disruption of the Asn414-Asp515 hydrogen bond on the surface of the protein and displacement of the N- and C-terminal flanking regions of LOV2 (404-546), both of which constitute a structural signal.

Structural Basis for Light-dependent Signaling in the Dimeric LOV Domain of the Photosensor YtvA

The photosensor YtvA binds flavin mononucleotide and regulates the general stress reaction in Bacillus subtilis in response to blue light illumination. It belongs to the family of light-oxygen-voltage (LOV) proteins that were first described in plant phototropins and form a subgroup of the Per-Arnt-Sim (PAS) superfamily. Here, we report the three-dimensional structure of the LOV domain of YtvA in its dark and light states. The protein assumes the global fold common to all PAS domains and dimerizes via a hydrophobic interface. Directly C-terminal to the core of the LOV domain, an α-helix extends into the solvent. Light absorption causes formation of a covalent bond between a conserved cysteine residue and atom C(4a) of the FMN ring, which triggers rearrangements throughout the LOV domain. Concomitantly, in the dark and light structures, the two subunits of the dimeric protein rotate relative to each other by 5°. This small quaternary structural change is presumably a component of the mechanism by which the activity of YtvA is regulated in response to light. In terms of both structure and signaling mechanism, YtvA differs from plant phototropins and more closely resembles prokaryotic heme-binding PAS domains.

Reevaluating the relationship between EPR spectra and enzyme structure for the iron–sulfur clusters in NADH:quinone oxidoreductase

NADH:quinone oxidoreductase (complex I) plays a pivotal role in cellular energy production. It employs a series of redox cofactors to couple electron transfer to the generation of a proton-motive force across the inner mitochondrial or bacterial cytoplasmic membrane. Complex I contains a noncovalently bound flavin mononucleotide at the active site for NADH oxidation and eight or nine iron-sulfur clusters to transfer electrons between the flavin and a quinone-binding site. Understanding the mechanism of complex I requires the properties of these clusters to be defined, both individually and as an ensemble. Most functional information on the clusters has been gained from EPR spectroscopy, but some clusters are not observed by EPR and attributing the observed signals to the structurally defined clusters is difficult. The current consensus picture relies on correlating the spectra from overexpressed subunits (containing one to four clusters) with those from intact complexes I. Here, we analyze spectra from the overexpressed NuoG subunit from Escherichia coli complex I and compare them with spectra from the intact enzyme. Consequently, we propose that EPR signals N4 and N5 have been misassigned: signal N4 is from NuoI (not NuoG) and signal N5 is from the conserved cysteine-ligated [4Fe-4S] cluster in NuoG (not from the cluster with a histidine ligand). The consequences of reassigning the EPR signals and their associated functional information on the free energy profile for electron transfer through complex I are discussed.

Steric Interactions Stabilize the Signaling State of the LOV2 Domain of Phototropin 1

Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.

Common conformational changes in flavodoxins induced by FMN and anion binding: The structure of Helicobacter pylori apoflavodoxin

Flavodoxins, noncovalent complexes between apoflavodoxins and flavin mononucleotide (FMN), are useful models to investigate the mechanism of protein/flavin recognition. In this respect, the only available crystal structure of an apoflavodoxin (that from Anabaena) showed a closed isoalloxazine pocket and the presence of a bound phosphate ion, which posed many questions on the recognition mechanism and on the potential physiological role exerted by phosphate ions. To address these issues we report here the X-ray structure of the apoflavodoxin from the pathogen Helicobacter pylori. The protein naturally lacks one of the conserved aromatic residues that close the isoalloxazine pocket in Anabaena, and the structure has been determined in a medium lacking phosphate. In spite of these significant differences, the isoallozaxine pocket in H. pylori apoflavodoxin appears also closed and a chloride ion is bound at a native-like FMN phosphate site. It seems thus that it is a general characteristic of apoflavodoxins to display closed, non-native, isoalloxazine binding sites together with native-like, rather promiscuous, phosphate binding sites that can bear other available small anions present in solution. In this respect, both binding energy hot spots of the apoflavodoxin/FMN complex are initially unavailable to FMN binding and the specific spot for FMN recognition may depend on the dynamics of the two candidate regions. Molecular dynamics simulations show that the isoalloxazine binding loops are intrinsically flexible at physiological temperatures, thus facilitating the intercalation of the cofactor, and that their mobility is modulated by the anion bound at the phosphate site.

Functional Expression of Mosquito NADPH-Cytochrome P450 Reductase in Escherichia coli

A complete NADPH-cytochrome P450 reductase (CPR) cDNA was isolated from Anopheles minimus Theobald mosquitoes by using reverse transcription-polymerase chain reaction-based methods. The complete cDNA contains 2,040 bp encoding the protein of 679 amino acids, with a calculated molecular mass of 77.3 kDa. The deduced amino acid sequence had a typical feature of CPR by possessing conserved domains involved in binding of flavin mononucleotide, flavin adenine dinucleotide, and NADPH cofactors. The complete CPR cDNA was expressed as 6x His-tagged fusion protein in both membrane and cytosolic fractions in Escherichia coli, both fractions contained NADPH-cytochrome c reducing activity. The membrane-bound form containing N-terminal membrane anchor was subjected to purification, and Km values were determined for NADPH and cytochrome c. The purified CPR enzyme was functionally active, as demonstrated by its ability to support CYP6AA3-mediated metabolism in the reconstituted reaction in vitro. Initial test suggested that CYP6AA3 could play a role in deltamethrin metabolism.

Functional Expression of Mosquito NADPH-Cytochrome P450 Reductase in <I>Escherichia coli</I>

A complete NADPH-cytochrome P450 reductase (CPR) cDNA was isolated from Anopheles minimus Theobald mosquitoes by using reverse transcription-polymerase chain reaction-based methods. The complete cDNA contains 2,040 bp encoding the protein of 679 amino acids, with a calculated molecular mass of 77.3 kDa The deduced amino acid sequence had a typical feature of CPR by possessing conserved domains involved in binding of flavin mononucleotide, flavin adenine dinucleotide, and NADPH cofactors. The complete CPR cDNA was expressed as 6x His-tagged fusion protein in both membrane and cytosolic fractions in Escherichia coli, both fractions contained NADPH-cytochrome c reducing activity. The membrane-bound form containing N-terminal membrane anchor was subjected to purification, and K, values were determined for NADPH and cytochrome c. The purified CPR enzyme was functionally active, as demonstrated by its ability to support CYP6AA3-mediated metabolism in the reconstituted reaction in vitro. Initial test suggested that CYP6AA3 could play a role in deltamethrin metabolism.

Ab Initio Quantum Chemical Investigation of the First Steps of the Photocycle of Phototropin: A Model Study¶

Abstract Phototropin is a blue light–activated photoreceptor that plays a dominant role in the phototropism of plants. The protein contains two subunits that bind flavin mononucleotide (FMN), which are responsible for the initial steps of the light-induced reaction. It has been proposed that the photoexcited flavin molecule adds a cysteine residue of the protein backbone, thus activating autophosphorylation of the enzyme. In this study, the electronic properties of several FMN-related compounds in different charge and spin states are characterized by means of ab initio quantum mechanical calculations. The model compounds serve as idealized model chromophores for phototropism. Reaction energies are estimated for simple model reactions, roughly representing the addition of a cysteine residue to the flavin molecule. Excitation energies were calculated with the help of time-dependent density functional theory. On the basis of these calculations we propose the following mechanism for the addition reaction: (1)...

Characterization of a Novel Bifunctional Dihydropteroate Synthase/Dihydropteroate Reductase Enzyme from Helicobacter pylori

Tetrahydrofolate is a ubiquitous C(1) carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl tRNA(fMet), which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence of Helicobacter pylori failed to reveal any sequence that encodes DHFR. Previous studies demonstrated that the H. pylori dihydropteroate synthase gene folP can complement an Escherichia coli strain in which folA and folM, encoding two distinct DHFRs, are deleted. It was also shown that H. pylori FolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated that H. pylori FolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzyme-bound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.

The LOV domain: a chromophore module servicing multiple photoreceptors

Three different families of blue-light receptors have been characterized from higher plants: three cryptochromes, two phototropins, and the three members of the ZTL/ADO family. Phototropins and the ZTL/ADO proteins have chromophore modules, designated LOV domains, that bind flavin mononucleotide and undergo formation of a C(4a) flavin-cysteinyl adduct. All contain the highly conserved amino acid motif GXNCRFLQ. Over 90 prokaryote proteins also contain LOV domains with this motif upstream from one of several different functional groups. All of these that have been investigated to date act as photoreceptors in vitro and form the adduct upon irradiation. Four members of the class LOV-histidine kinase, one from a plant pathogen (Pseudomonas syringae), one from an animal pathogen Brucella melitensis), and two from a marine bacterium (Erythrobacter litoralis) respectively, mediate light-activated histidine phosphorylation. Decay of the adduct in darkness after a blue light pulse coincides with loss of the capacity for phosphorylation upon addition of ATP. At present, the biological role(s) of these light-sensitive proteins is under investigation.

A LOV Story: The Signaling State of the Phot1 LOV2 Photocycle Involves Chromophore-Triggered Protein Structure Relaxation, As Probed by Far-UV Time-Resolved Optical Rotatory Dispersion Spectroscopy

Light-, oxygen-, or voltage-regulated (LOV1 and LOV2) domains bind flavin mononucleotide (FMN) and activate the phototropism photoreceptors phototropin 1 (phot1) and phototropin 2 (phot2) by using energy from absorbed blue light. Upon absorption of blue light, chromophore and protein conformational changes trigger the kinase domain for subsequent autophosphorylation and presumed downstream signal transduction. To date, the light-induced photocycle of the phot1 LOV2 protein is known to involve formation of a triplet flavin mononucleotide (FMN) chromophore followed by the appearance of a FMN adduct within 4 micros [Swartz, T. E., Corchnoy, S. B., Christie, J. M., Lewis, J. W., Szundi, I., Briggs, W. R., and Bogomolni, R. A. (2001) J. Biol. Chem. 276, 36493-36500] before thermal decay back to the dark state. To probe the mechanism by which the blue light information is relayed from the chromophore to the protein, nanosecond time-resolved optical rotatory dispersion (TRORD) spectroscopy, which is a direct probe of global secondary structure, was used to study the phot1 LOV2 protein in the far-UV region. These TRORD experiments reveal a previously unobserved intermediate species (tau approximately 90 micros) that is characterized by a FMN adduct chromophore and partially unfolded secondary structure (LOV390(S2)). This intermediate appears shortly after the formation of the FMN adduct. For LOV2, formation of a long-lived species that is ready to interact with a receptor domain for downstream signaling is much faster by comparison with formation of a similar species in other light-sensing proteins.

BluB cannibalizes flavin to form the lower ligand of vitamin B12

Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms. The biosynthesis of the lower ligand of vitamin B(12), 5,6-dimethylbenzimidazole (DMB), is poorly understood. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and D-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease.