fluorescence and optical characteristics of reduced flavins and flavoproteins

7/30/2019 Fluorescence and Optical Characteristics of Reduced Flavins and Flavoproteins

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361EDUCED FLAVINSIn contrast, reduced flavins and flavoenzyrnes (F1M1) have a ratheratypical and variable absorption above 300 nm and have long been considered to be nonfluorescent." This. and the technical problems derivingfrom the sensitivity of reduced flavins toward oxygen. probably havediscouraged similar detailed investigations. An understanding of reducedflavins, however, is even more relevant than an understanding of oxidizedflavins, In fact, in add it ion to being the counterpart of Flux in redoxreactions. reduced flavins activate molecular oxygen inflavin-dependent

oxidases, in monooxygenases, and in bacterialluciferase. 7,8

uction ProceduresDithionite. This agent can be either a one- or a two-electron reducingagent. ll ,12 Its potential of -0.66 mVB is sufficiently low for the reduction

of most flavoproteins: some of those which we have reduced by thismethod are l is ted in the tab le . Dithionite i tsel f has a rather s trong absorpt ion at 315 nm (E = 5500-8000)11; therefore. the addition of excesses

Methods of Prepara tion of Reduced Flavins an d FlavoproteinsBasically any reducing agent of suitable potential will reduce freeoxidized flavins (E 0' = 200 mV) to the 1.5-dihydro lever. In the case ofsome flavoproteins, kinetic barriers. or the low potential for reduction of

the semiquinone to the dihydro form, can render fun reduction verydifficult to attain." The choice of the reducing agents willdepend on thescope of the investigation planned; for this reason. the most frequentlyemployed methods will be briefly discussed below. Whenthe reductionis carried out to obtain reduce flavins for spectroscopic measurements.Tunberg-type cuvettes are best employed. An apparatus suitable for suchexperiments has been described by Foust et al." This set-up can bemodified most conveniently by use of an air-tight Hamilton syringe gluedinto an appropriate ground-glass joint. instead of the original'" titrationglass burette.

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[51] Fluorescence an d Optical Characteristics ofReduced Flavins an d FlavoproteinsBy SANDRO GHISLA

Flavins and flavoproteins in their oxidized state (Flox) are. as the nameindicates. typically yellow, with the absorption spectrum in the visibleand near-ultraviolet (UV) range being characterized by two well-resolvedbands centered around 450 and 370 nm. Thi s chromophore, free in solut ion. exhib it s a s trong f luorescence tha t is maximal around 520 nm:when the chromophore is bound to flavoproteins, however. its emissioncan be fully quenched. These favorable properties have promoted manydetailed spectroscopic investigations dealing with theoretical aspects.with model compounds, and with flavoenzymes':": see also this volume[50].I Several articles inT. P. Singer. ed.. "Flavinsand Flavoproteins." Elsevier. Amsterdam.1976.2 P. S. Song. in "Flavins and Flavoproteins," (H. Kamin. ed.). p. 37. Univ. Park Press,Baltimore. Maryland, 1971.3 M. Sun, T. A. Moore . and P. S. Song. J. Am. Chem. Soc. 94. 1930(1972). J. L. Fox. S. P. Laberge, K. Nishimoto.and L. S. Forster, Biochim, Biophys, Ac({/ 109.626 (1965).s F . Mull er , S . G. Mayhew. and V. Massey, Biochemistry 12.4654 (1973). and articlescited therein.

S. Ghisla. V. Massey, J. M. Lhoste, and S. G. Mayhew, BiochemistrY 13.589 (1974).7 V. Massey and P. Hemmerich. in "The Enzymes" (P. D. Boyer, ed.). 3rd ed.. Vol . 12.p. 191.Academic Press . New York, 1975. H. Bright and D. J. T. Porter, in "The Enzymes" (P. D. Boyer. ed.l. 3rd ed.. Vol . 12.p. 421. Academic Press. New York, 1975. S. G. Mayhew. Eur. J. Biochem, (in press).

10 G. P. F ous t. B. D. Burleigh, S. G. Mayhew. C. H. Williams. and V. Massey. Anal.Biochem, 27,530 (1969).

II S. G. Mayhew and V. Massey, Biochim. Biophvs. Acta 315,181 (1973112 D. O. Lambeth and G. Palmer. J. Bioi. Chern. 248.6095 (1973).

First publ. in: Methods in Enzymology 66, (1980), pp. 360-373

Konstanzer Online-Publikations-System (KOPS)URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/6823/

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-68233

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FIG. I. Absorption spectra of flavin mononucleotide (FMN) in aqueous solution in thoxidized and reduced state and in the neutral and anionic state. FMN", in0.033N NaOJ(_ ' ' '_) ; FMN"x at pH 5 in 0.09 M c it ra te buf fe r (.... ) ; FMN..d at pH 8.5 in 0.1 lpyrophosphate buffer (---); FMN red at pH 5 i n 0.09 M citrate buffer (---). The redu.tions were achieved by illumination in the presence of EDTA. Adapted with permissicfrom S. Ghisla . V. Massey. J. M. Lhoste, and S. G. Mayhew. Biochemistry B, 589 (1974Copyright by the American Chemical Society.

state, 1.5-dihydroflavinsare "antiaromatic" (8 7Telectrons inthe pyrazinring), while in a "bent conformation" the tricyclic resonance of thisoalloxazine system might be restricted. Thus, depending on the degreof "bending" around the N(S)-N(lO) axis, the al lowedness of sever;electronic transitions might be greatly influenced, and resultin absorpticspectra of very different shapes.4a .5-Dihydroflavins

The near-U'V absorption spectrum of the 4a.S-dihydrofla\'in chrorophore is similar. in its appearance. to that of its 1.5-dihydroisomer. bits shape is even more dependent on temperature and solvent (Fig. 3).particular. substituents which have an effect on the internal mobilitythe molecule were found to cause major spectral changes on the chrorophore free in solut ion," and also when bound to various apoflavopr

- IC H 3 X C ~ ~ ' j O . io 54& N'H 1CH 3 N" II 0 I40,5-0ihydroflovin ;, 5 - OihydrofIovin

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Structures derived from I,&-, 1,8-, 1,3-, lOa-I dihydroflavin specieshave been discussed at various times,Z3 in particular in connection withthe structure of possible adducts of reduced flavin and oxygen. C(4)carbonyl reduced dihydroflavins." and those which are derived frombenzene ring hydrogenation," do not play any biological role. and willthus be omitted from the discussion.J.5-DihydrojlavinsThe I,S-Flred flavin chromophore is characterized by a rather structureless absorption in the visible and near-UV range, the shape of whichis strongly influenced by temperature, the ionization state of the flavin,the p rope rti es of the solvent, and by the pr esence of substituents in

particular at positions N(l) and N(S). The ionization at position N(I) (pK= 6.S), causes a =SO-nmhypsochromic shift of the longest wavelengthmaximum in aqueous media (Fig. I) . In ethanol and at ambient temperature, the effect of ionization is less pronounced (Fig. 2). While thesespecies are nonfluorescent in solution and at ambient temperature, theirincorporation in a glass matrix causes a white-bluish fluorescence toappear (Amax = Soo nm) which is the mirror image of the absorptionspectrum recorded under the same conditions (Fig. 2).6 Furthermore,under the conditions of Fig. 2, the absorption spectra now appear muchbetter resolved and indicate that the I,S-dihydroflavin chromophore hasmain 7T - 7T* transitions centered at =400, =3S0, and =29S nm. Lowtemperature spectra of N(1)- or N(S)-substituted dihydroflavins confirmthis attribution.IThe peculiar variability of the absorption and fluorescence spectra ofthe I,S-dihydroflavin chromophore probably originates in its "butterflyshaped" structure.s-v This molecule can undergo a series of vibrations[e.g., N(S) and N(lO) pyramidal nitrogen inversions, N(S)-NOO) ringinversion}" leading to different conformational states. In their coplanar.. P. Hemmerich and A. Wessiak . Flavins Flavoproelns, Proc. Int. Symp., Sth, 1975. p.9 (1976). L . Tauscher . S. Ghisla, and P. Hemmerich. Helv, Chim. Acta 56,630 (1973).

t A'366 FLAVINS AND DERIVATIVES.) [51] [et

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370 FLAVINS AND DERIVATIC/ [51] .51] U,DUCED FLAVINS 371

. .. P. Kierkegaard, R. Norrestam, P. Werner. I. Csoregh, M. von Glehn, R. Karlsson. M.Leijonrnark, O. Ronnquist, B. Stensland, O. Tillberg, and L. Torbjornsson, in "Flaviusand Flavoproteins" (H. Kamin, ed.),p. I. Univ. Park Press. Baltimore. Maryland. 1971.21 K. H. Dudley. A. Ehrenberg, P. Hemmerich. and F. Mii ller, Hell'. Chim. Acta 47. 1354

(1%4).B. Entsch. D. Ballou. and V. Massey J. BioI. Chern, 251,2550 (1976)... W. Hastings. C. Balny. and P. Douzou. Flavins Flavoproteins, Proc, Int. Symp . Sth,

/975 p. 53(1976).

FIG. 4. Absorption. fluorescence emission. and fluorescenceexcitation spectraoflactateoxidase . 3 x 10- M in 0.01 M imidazole-HCl buffer (pH 7.0) at 25. Absorption spectrumof the oxidized enzyme (_._. ) : absorption of the enzyme reduced with t-lactate , after thedecay of the ini tial ly formed. nonfluorescent complex (---); fluorescence emissionAexr ,.. non 360 nm) (----); fluorescence excitation spectrum (A.m,.. ton 507 nm) of the reducedenzyme (.... ). The oxidized enzyme does not show detectable fluorescence. Adapted withpermissionfrom S. Ghisla. V. Massey. J . M. Lhoste, and S. G. Mayhew. Biochemistry 13.589 (1974). Copyright by the American Chemical Society.

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zymes in order to study their spectral properties (see the table). 25.30 Theeffects observed upon complexat ion with the protein are even morepronounced than those observed within 1,5-dihydroflavoproteins. A typical case is illustrated in Fig. 6, where upon binding to the apoenzyme ofn-amino acid oxidase the absorption of 4a-hydroxy-S-ethyl-FAD (compare to Fig. 3) is modified to a single-banded spectrum in the near-UVrange (Fig. 6), and the extinction coefficient is increased by =30%. Aqualitatively similar effect is observed with the 4a.5-dihydroflavin modelof Fig. 3 upon freezing into a glassy matrix.ao S. Ghi sl a, H. Ogata . V. Massey, A. Schonbrunn. R. H. Abeles. and C. T. Walsh.

Biochemistry IS. 1791 (1976).

FIG. 5. Absorption. fluorescence emission. and fluorescence excitation spectra of flavodoxin from P. elsdenii. 2 x 10->M. in 0.1 M pyrophosphate buffer (pH 8.5) at 2 ~ 0 .Absorption of the oxidized enzyme (_._. ) ; absorption spectrum of the enzyme reducedwith dithionite in the apparatus described by Fourst e t a l. (---I [G. P. Fo ust . B. D.Burleigh. S. G. Mayhew. C. H. Williams. and V. Massey. Anal. Biochem. 27.530 (I969));fluorescence emission (Am tlon 368nm) (----); fluorescence excitation spectrum (A.m, .. ten530 nm) of the reduced enzyme (.. ..) . The oxidized enzyme is devoid of fluorescence.Adapted with permission from S. Ghisla. V. Massey. J . M. Lhoste, and S. G. Mayhew.Biochemistry 13.589 (1974). Copyright by the American Chemical Society.

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, " - - - - - - 372 FLAVINS AND DERIVAyf . . [51) ~ ~ 2 ) TIME-RESOLVED FLUORESCENCE ON FLAVINS 373

FIG. 6. Absorption and fluorescence emrssion spectrum of 4a-hydroxy-."-ethyl-FADbound to o-aminoacid apooxidase. The complex was prepared by incubation for 10min at0" of 0.5 ml 10-4 M n-amino acid apooxidase with IO-fold excess of the modified FAD in0.05 M phosphate buffer. pH 7.5." The protein was separated from excess coenzyme bychromatography over a short Sephadex G-25equilibrated with the same buffer. and at 0-4. Curve (---), absorption spectrum. and curve (_._. ) , fluorescence emission spectrum of the compl ex . 2 x 10-' M. The excitation wavelength was 375 nm. The excitationspectrum. recorded with A.m'..'on = 500nm, closely foUowsthe absorption spectrum. Curve(.... ) shows the spectrumof the oxidized enzymeunder the same conditionsfor comparison.

Note that spectral effects such as those shown in Fig. 6 are mostprobably not due to chemica l modif icat ion of the 4a.5-dihydroflavinchromophore upon binding to the apoprotein, as has been observed incertain cases." In fact the 4a-hydroxy-5-ethyl-4a,5-dihydro chromophorecould be recovered intact after such experiments upon denaturation ofthe protein." We observed recently that, in contrast to common beliefs,reduced flavoproteins can be fluorescent.6 Among the examples listed onthe table, roughly one-half were found to exhibit measurable fluorescenceemission in the range 460-530 nm. This was taken as an indication" thatthe reduced flavin coenzyme might be pl aced in a rigid environment atthe enzyme-active center. Similarly, at least some of the inversion processes occurring in solution" might be slowed down to such an extentthat they do not give a major contribution to radiationless energy transfer . .Supporting this hypothesis is the fact that in several cases introductionof substituents, which will enhance r igidity of the reduced flavin, has

AcknowledgmentI wi sh to thank Drs. S. G. Mayhew and P. Hemmerrich for manyhelpful suggestions.

at V. Massey and S. Ghisla. Proc. IOlh FEBS Meet. p. 145(1975)... S. Ghisla and V. Massey. in "Mechanisms of Redox Enzymes" (R. Ondarza and T. P.Singer. eds.). Am. Elsevier, New York (in press).

been found to increase the fluorescence yield (see the table). :n.aJ Nocorrelation between occurrence of fluorescence and function of the flavoenzyme has been found. On the other hand, a general trend can beseen where those enzymes tha t are fluorescent in the oxidized s ta te arenot in the reduced state, and vice versa.Finally, we wish to emphasize the potential usefulness of fluorescencestudies for the investigation of flavoenzyme reaction mechanisms, wheretransient intermediates having the reduced flavin chromophore might beaccessible to characterization. In fact, recently covalent intermediatesoccurring during the reaction of lactate oxidase with glycolate could bedemonstrated by taking advantage of their strong fluorescence emissionat 465 nm.:U .32

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fluorescence and optical characteristics of reduced flavins and flavoproteins

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