Covalent Intermediates in Flavin-sensitized Photodehydrogenation and Photodecarboxylation

Knappe, Wolfgang‐R.; Hemmerich, Peter

doi:10.1515/znb-1972-0907

Cited by 18 publications

(7 citation statements)

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“…There is presumably sufficient bound flavin in the particulate fraction to suffice without further addition. That the flavin is the photoreceptor in the present work is strongly suggested by the observation (Brain, unpublished) that phenylacetic acid, known to form a fairly stable covalent adduct with the triplet state of flavins (11), completely blocks the Cyt photoreduction in the resuspended 2OKP from Neurospora.…”

Section: Methodssupporting

confidence: 60%

Blue light-induced Absorbance Changes in Membrane Fractions from Corn and Neurospora

Brain¹,

Freeberg²,

Weiss³

et al. 1977

Plant Physiol.

113

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Blue light-induced absorbance changes were measured from differentially centrifuged membrane fractions from dark-grown coleoptiles of Zea mays L., and mycelia from an albino mutant of Neurospora crassa Actinic irradiation caused changes in absorbance consistent with a flavinmediated reduction of a b-type cytochrome. Both corn and Neurospora showed similar light-minus-dark difference spectra, dose response curves, and kinetics of dark recovery after irradiation. The photoreducible cytochrome system from Neurospora showed the same distribution as the activity of a sodium-stimulated adenosine triphosphatase, thought to be a plasma membrane marker, in differential centrifugation experiments. The fraction showing the absorbance change did not co-sediment with the mitochondria, nor with the endoplasmic reticulum. Comparison of absorption spectra of fully oxidized, partially reduced, and fully reduced preparations showed that approximately a 30% reduction of the cytochromes involved with the process was needed to obtain the lightinduced absorbance changes.There is currently a substantial body of literature concerning the effects of blue light on biological processes in higher plants and fungi. Only recently has any real progress been made toward the identification of the blue light photoreceptor associated with these effects (see ref. 6). Poff and Butler (16) ation in that report was the separation of soluble from pelletable components. Mufioz and Butler (15) did report light-inducible Cyt reduction in soluble extracts of Neurospora, but they studied supernatant rather than pelletable activity, observed reduction both of Cyt b and c, and had to add FMN or FAD in order to observe the reaction. It seems likely that they were studying the same phenomenon as Schmidt and Butler (20) who found photoreduction of Cyt c in the presence of added flavins in a model system. Widell and Bjorn (24) have reported light-induced A changes in another higher plant system, intact wheat coleoptiles, but the responses measured were not the same as those monitored by Briggs (5) or Munioz and Butler (15).The purposes of the study reported here were: (a) to isolate and characterize partially purified membrane fractions of Neurospora with a photoreduction system similar to that found in intact mycelia by Munoz and Butler; (b) to study further the blue light-induced A changes found in corn membrane fractions and compare them to similar changes found in Neurospora; and (c) to determine qualitatively the redox state necessary for observation of these changes. A preliminary account of these studies has appeared elsewhere (3, 7). To prepare the mycelia, approximately 70-ml liquid suspensions were made of the 7-day-old agar cultures, and added to a 2,000-ml Erlenmeyer flask, filled with 1,000 ml of liquid medium (the same as solid medium except dextrose instead of sucrose, and no agar). The cultures were grown aerobically in the dark at 30 C for 24 hr at 250 rpm in an incubating rotary shaker (New Brunswick Scientific,. MATERIALS AND METHODSA...

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Section: Methodssupporting

confidence: 60%

Blue light-induced Absorbance Changes in Membrane Fractions from Corn and Neurospora

Brain¹,

Freeberg²,

Weiss³

et al. 1977

Plant Physiol.

113

View full text Add to dashboard Cite

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“…The putative signaling state of PYP stores only about 20% of the E 00 energy due to a partial light‐induced unfolding (105). The high value of E 390 in LOV proteins drives the completion of the photocycle and the return to the dark state that would otherwise be virtually impossible given the intrinsic stability of photochemically formed C(4)a‐alkylated flavin adducts in solution (106). This inherent stability has been also confirmed by theoretical calculations (107), that provide a negative energy content for the FMN‐C(4)a‐Cys adduct (−53.55 kJ mol −1 ).…”

Section: Small Photosensing Units: the Lov And Bluf Domainsmentioning

confidence: 99%

Flavin‐based Blue‐light Photosensors: A Photobiophysics Update

Losi¹

2007

Photochem & Photobiology

190

205

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This review deals with the biophysical aspects of flavin-based photosensors, comprising cryptochromes, LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using FAD) proteins. Special emphasis is given to structural issues, photocycle quantum yields and energetics, mechanism of the lighttriggered reactions, early stages in signal transduction and oligomeric states of the light sensing protein modules. For BLUF and LOV domains important parallels are emerging, despite their different a ⁄ b fold arrangement, whereas there is increasing evidence for a mechanicistic and functional splitting of the cryptochrome family.

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“…Nonphotochemically formed flavin C(4a)-thiol adducts are catalytic intermediates in flavoenzymes with redox active disulfides (Williams, 1992) and possible intermediates in the biosynthesis of oligomeric enzymes (Eschenbrenner et al, 2001). The photochemical formation of C(4a)-thiol adducts in solution was first described by Knappe and Hemmerich using sulfur compounds as substrates and suggested to proceed via the decay of the excited flavin triplet state (Knappe and Hemmerich, 1972). Accordingly, transient spectroscopic measurements of phot-LOV domains, have revealed that the photocycle comprises a red-shifted transient species, LOV 715 (appearing within 20 ns, also referred to as LOV 660 ), spectroscopically similar to the FMN triplet excited state Kottke et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Recording of Blue Light-Induced Energy and Volume Changes within the Wild-Type and Mutated Phot-LOV1 Domain from Chlamydomonas reinhardtii

Losi

Kottke

Hegemann

2004

Biophysical Journal

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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.

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Covalent Intermediates in Flavin-sensitized Photodehydrogenation and Photodecarboxylation

Cited by 18 publications

References 0 publications

Blue light-induced Absorbance Changes in Membrane Fractions from Corn and Neurospora

Blue light-induced Absorbance Changes in Membrane Fractions from Corn and Neurospora

Flavin‐based Blue‐light Photosensors: A Photobiophysics Update

Recording of Blue Light-Induced Energy and Volume Changes within the Wild-Type and Mutated Phot-LOV1 Domain from Chlamydomonas reinhardtii

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