Flavocytochromes c: transient kinetics of photoreduction by flavin analogues

Kinetics of reduction of phototrophic bacterial flavocytochromes c by exogenous flavin semiquinones and fully reduced flavins generated by laser flash photolysis have been studied. The mechanisms of reduction of Chromatium and Chlorobium flavocytochromes c are more similar to one another than previously thought. Neither protein is very reactive with neutral flavin semiquinones (k less than 10(7) M-1 s-1), and the reactions with fully reduced flavins are slower than expected on the basis of comparison with other electron-transfer proteins of similar redox potentials. Deazaflavin radical is reactive with the flavocytochromes c by virtue of its low redox potential, but this reaction is also slower than expected on the basis of comparison with other electron-transfer proteins. These experiments indicate that the active site for reduction of flavocytochrome c is relatively buried and probably inaccessible to solvent. Fully reduced FMN does not show an ionic strength effect in its reaction with flavocytochrome c, which demonstrates that the active site is uncharged. Sulfite, which forms an adduct with protein-bound FAD, partially blocks heme reduction. This shows that heme is reduced via the FAD. The rate constant for intramolecular electron transfer between FAD and heme must be on the order of 10(4) s-1 or larger.     

Probing the reactivity of different forms of azurin by flavin photoreduction

The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild-type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5-deazariboflavin and 8α-imidazolyl-(N-propylyl)-amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co-ordination shell was filled with imidazole. 8α-Imidazolyl-(N-propylyl)-amino riboflavin appears one to two orders less efficient as a photo-flash reductant than 5-deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5-fold lower than the WT reactivity (second-order rate constants of 51 ± 2 × 10(7) m(-1) ·s(-1) versus 21 ± 1 × 10(7) m(-1) ·s(-1) ), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 10(7) m(-1) ·s(-1) ). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database Structural data are available in the Protein Data Bank under the accession number 3N2J.     

Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii

Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.     

Human and Drosophila cryptochromes are light activated by flavin photoreduction in living cells

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non-light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.     

Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.     

Irreversible denaturation mapping of a pyrimidine-rich domain of a complex satellite DNA

The highly complex G + C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis has been studied by denaturation mapping. Following digestion of the satellite with EndoR.Eco RI, the major 2.07-kilobase pair (kbp) basic repeating unit and a minor 4.14-kbp fragment were exposed to 254 nm light in the presence of silver ions, conditions which resulted in essentially irreversible denaturation of regions rich in adjacent pyrimidines by the formation of pyrimidine dimers. The positions and sizes of the denatured regions were determined in electron micrographs of partially denatured 2.07-kbp and 4.14-kbp fragments spread in the presence of formamide. After 15 min exposure to UV, 90% of the 2.07-kbp fragments had a denaturation bubble averaging 0.17 kbp centered around one-third (0.64 kbp) the total length; 20% exhibited another in the region from 1.8 kbp to 2.07 kbp. Similarly, about 90% of the 4.14-kbp fragments had denatured regions centered at 0.64 kbp and 2.75 kbp and 20% of the fragments had denaturation bubbles in regions centered at 1.92 kbp and 3.9 kbp. The positions of the denaturation bubbles in the 4.14-kbp fragments support restriction enzyme mapping evidence that it is a dimer of the 2.07-kbp fragment arranged head to tail. Sequencing data show that the predominant sequence of a 0.29-kbp region centered around 0.64 kbp in the basic repeat unit is 49% A + T and that 42% of the bases are adjacent TTs and CTs capable of dimerization under the conditions used.     

Modeling the dynamics of a mutated stem-loop in the SL1 domain of HIV-1Lai genomic RNA by 1H-NOESY spectra

The cross-peaks of ¹H-NOESY spectra at different time delays are compared to a mode-coupling diffusion (MCD) calculation, including the evaluation of the full ¹H relaxation matrix, in the case of a 23 nucleotide fragment of the stem-loop SL1 domain of HIV-1_Lai genomic RNA mutated in a single position. The MCD theory gives significant agreement with ¹H relaxation experiments enabling a thorough understanding of the differential local dynamics along the sequence and particularly of the dynamics of nucleotides in the stem and in the loop. The differential dynamics of this hairpin structure is important in directing the dimerization of the retroviral genome, a fundamental step in the infectious process. The demonstration of a reliable use of time dependent NOE cross-peaks, largely available from NMR solution structure determination, coupled to MCD analysis, to probe the local dynamics of biological macromolecules, is a result of general interest of this paper.     

Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii

The “Phot” protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 μs with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.     

Antibody-induced down-regulation of a mutated insulin receptor lacking an intact cytoplasmic domain

Insulin receptor down-regulation was studied in various Chinese hamster ovary (CHO) cell lines expressing transfected human insulin receptor cDNAs. In addition to a cell line expressing the normal receptor (CHO.T line), three lines expressing mutated receptors were studied: the CHO.T-t line, which expresses a receptor with a degraded cytoplasmic domain due to the removal of the C-terminal 112 amino acids, and the CHO.YF1 and CHO.YF3 lines, in which important autophosphorylation sites of the receptor kinase (tyrosines-1162 and -1163) have been replaced by phenylalanine. A monoclonal anti-receptor antibody, but not insulin itself, was found to down-regulate cell surface receptor levels in all four cell lines by 60-80% after 18-h treatment at 37 degrees C. Down-regulation of the CHO.T and CHO.T-t receptors occurred at similar antibody concentrations and with a similar time course, although the maximum level of CHO.T-t down-regulation (60%) was generally lower than the level of CHO.T down-regulation (80%). Pulse-chase labeling of these two cell types with [35S]methionine revealed that antibody treatment of both CHO.T and CHO.T-t cells resulted in a similar increase in the rate of degradation of mature receptor subunits. These results indicate that antibody-induced down-regulation of the insulin receptor in these cells can occur in the absence of various autophosphorylation sites of the receptor and that the mechanism of antibody-induced down-regulation is different from that for insulin.     

Functional expression of Phanerochaete chrysosporium cellobiose dehydrogenase flavin domain in Escherichia coli

Cellobiose dehydrogenase (CDH; EC 1.1.99.18) is an extracellular glycosylated protein composed of two distinct domains, a C-terminal catalytic flavin domain and an N-terminal cytochrome-b-type heme domain, which transfers electrons from the flavin domain to external electron acceptors. The soluble flavin domain of the Phanerochaete chrysosporium CDH was successfully expressed in Escherichia coli. The enzyme showed dye-mediated CDH activity higher than that of the complete CDH, composed of flavin domain and heme domain, prepared using Pichia pastoris as the host microorganism. The ability to conveniently express the recombinant CDH flavin domain in E. coli provides great opportunities for the molecular engineering of the catalytic properties of CDH.     

Flavin-photosensitized oxidation of reduced c-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin-cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.     

Schizosaccharomyces pombe encodes a mutated AP endonuclease 1

Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.     

Induction of skin fibrosis in mice expressing a mutated fibrillin-1 gene

BACKGROUND: Tight skin mice (TSK) bear a mutated Fibrillin-1 (Fbn-1) gene. Genetic studies show that the TSK mutation is closely associated with the Fbn-1 locus (0-0.7 cM). A previous study showed two recombinants between the Fbn-1 locus and the TSK mutation. TSK mutation and mutated Fbn-1 gene cosegregate in F1 mice. MATERIALS AND METHODS: To elucidate the role of the mutated Fbn-1 gene in occurrence of TSK syndrome, we generated transgenic (Tg) mice expressing mutated Fbn-1 gene. In another set of experiments, we injected normal mice after birth with a plasmid bearing mutated Fbn-1 gene (pdFbn-1). RESULTS: Our results demonstrate that the pdFbn-1 Tg mice developed permanent cutaneous hyperplasia that was permanent. In mice injected as newborns with a plasmid bearing the sense pdFbn-1 gene, cutaneous hyperplasia was transient. In contrast to TSK mice, neither Tg nor mice injected with plasmid developed lung emphysema. The pdFbn-1 Tg and TSK mice spontaneously produced anti-topoisomerase I and anti-Fbn- antibodies, as do humans afflicted by scleroderma; whereas, those injected with a plasmid containing the pdFbn-1 gene produced only anti-Fbn-1 autoantibodies. CONCLUSIONS: The results suggest that, although cutaneous hyperplasia is due to mutated Fbn-1 gene, the TSK syndrome may be multifactorial.     

Heterogeneous environment of the S-H group of Cys966 near the flavin chromophore in the LOV2 domain of Adiantum neochrome1

The primary photochemistry of the blue-light sensor protein, phototropin, is adduct formation between the C4a atom of the flavin mononucleotide (FMN) chromophore and a nearby, reactive cysteine (Cys966), following decay of the triplet excited state of FMN. The distance between the C4a position of FMN and the sulfur atom of Cys966 is 4.2 A in the LOV2 domain of Adiantum neochrome 1 (neo1-LOV2), a fusion protein of phototropin containing the phytochrome chromophoric domain. We previously reported the presence of an unreactive fraction in neo1-LOV2 at low temperatures, which presumably originated from the heterogeneous environment of Cys966 [Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191]. The present study showed that (i) 28% forms an adduct at 77 K (state I), (ii) 50% forms an adduct at 150 K but not at 77 K (state II), and (iii) 22% does not form an adduct at 150 K (state III). By Fourier transform infrared (FTIR) spectroscopy, we observed the S-H stretching frequencies at 2570 and 2562 cm-1 for state I and at 2563 cm-1 for state II, suggesting that the microenvironment of the S-H group of Cys966 determines the reactivity at low temperatures. Adduct formation is more efficient for state I than for states II and III. Molecular dynamics simulation strongly suggests that the observed multiple structures originate from the isomeric forms of Cys966. We thus concluded that there are multiple local structures of FMN and cysteine in neo1-LOV2, each of which is thermally converted by protein fluctuation at physiological temperatures.     

Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro

DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P.F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (KA approximately 10(9) M-1) of the enzyme. The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.     

Pore properties of rat brain II sodium channels mutated in the selectivity filter domain

Ion selectivity of voltage-activated sodium channels is determined by amino-acid residues in the pore regions of all four homologous repeats. The major determinants are the residues DEKA (for repeats I-IV) which form a putative ring structure in the pore; the homologous structure in Ca-channels consists of EEEE. By combining site-directed mutagenesis of a non-inactivating form of the rat brain sodium channel II with electrophysiological methods, we attempted to quantify the importance of charge, size, and side-chain position of the amino-acid residues within this ring structure on channel properties such as monovalent cation selectivity, single-channel conductance, permeation and selectivity of divalent cations, and channel block by extracellular Ca²⁺ and tetrodotoxin (TTX). In all mutant channels tested, even those with the same net charge in the ring structure as the wild type, the selectivity for Na⁺ and Li⁺ over K⁺, Rb⁺, Cs⁺, and NH₄ ⁺ was significantly reduced. The changes in charge did not correlate in a simple fashion with the single-channel conductances. Permeation of divalent ions (Ca²⁺, Ba²⁺, Sr²⁺, Mg²⁺, Mn²⁺) was introduced by some of the mutations. The IC₅₀ values for the Ca²⁺ block of Na⁺ currents decreased exponentially with increasing net negative charge of the selectivity ring. The sensitivity towards channel block by TTX was reduced in all investigated mutants. Mutations in repeat IV are an exception as they caused smaller effects on all investigated channel properties compared with the other repeats. Received: 24 July 1996 / Accepted: 12 September 1996     

Recording of blue light-induced energy and volume changes within the wild-type and mutated phot-LOV1 domain from Chlamydomonas reinhardtii

The time-resolved thermodynamics of the flavin mononucleotide (FMN)-binding LOV1 domain of Chlamydomonas reinhardtii phot (phototropin homolog) was studied by means of laser-induced optoacoustic spectroscopy. In the wild-type protein the early red-shifted intermediate LOV(715) exhibits a small volume contraction, DeltaV(715) = -1.50 ml/mol, with respect to the parent state. LOV(715) decays within few micro s into the covalent FMN-Cys-57 adduct LOV(390), that shows a larger contraction, DeltaV(390) = -8.8 ml/mol, suggesting a loss of entropy and conformational flexibility. The high energy content of LOV(390), E(390) = 180 kJ/mol, ensures the driving force for the completion of the photocycle and points to a strained photoreceptor conformation. In the LOV-C57S mutated protein the photoadduct is not formed and DeltaV(390) is undetected. Large effects on the measured DeltaVs are observed in the photochemically competent R58K and R58K/D31Q mutated proteins, with DeltaV(390) = -2.0 and -1.9 ml/mol, respectively, and DeltaV(715) approximately 0. The D31Q and D31N substitutions exhibit smaller but well-detectable effects. These results show that the photo-induced volume changes involve the protein region comprising Arg-58, which tightly interacts with the FMN phosphate group.     

Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase

The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.