A SPECTRAL-LUMINESCENCE STUDY OF THE

NADPH: QUINONE OXIDOREDUCTASE ENZYME

B. A. Tatarinov, S. D. Speranskii, and A. I. Komyak

UDC 535.34./35:577.158

The coordinated application of spec t ra l methods for the study of biocatalytic sys tems permits a more complete charac te r iza t ion of these sys tems as well as of their behavior during the course of react ions . At present , about 50 flavoproteins f f lav in- -pro te in complexes), which catalyze redox react ions in biochemical p rocesses , are known [1]~ Recently, the mitochondrial NADPH: quinone oxidoreductase enzyme (EC 1.6.99.2), which is a member of the f lavoprotein c lass , was obtained for the f irst t ime [2]. The flavin components of flavoproteins part icipate in the hydrogen atom t rans fe r f rom a donor to an acceptor during the course of a r e - action. The specific spec t ra l - luminescent proper t ies of flavins permit us to study the mechanism of the ca ta - lysis performed by the enzyme. Only three flavin der ivat ives , namely, flavin adenine dinucleotide, flavin mononucleotide, and riboflavin, have been found as components of f lavoproteins. Titovets et al. [3], who iso- lated this enzyme, determined by biochemical methods that NADPH: quinone oxidoreduetase contains flavin adenine dinucleotide.

The present work was devoted to a spect roscopic study of the NADPH: quinone oxidase enzyme with the aim of clarifying the s t ruc ture and composition, as well as the nature of the binding and environment of the flavin, in this f!avoprotein.

The enzyme was isolated by the procedure described by Titovets et al. [3] and then subjected to lyo- philic drying and stored in sealed ampules. The puri ty of the prepara t ion was checked using e lec t rophores is in polyacrylamide gel. Samples of flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleo- tide (NADH2) , riboflavin, and papain were obtained f rom Reanal and used without further purification. A Spe- cord UV VIS spec t rophotometer , Fica-55 spec t rof luor imeter , and Spectropol-1 spect rophotometer with an at tachment for record ing c i rcu la r d ichro i sm spec t ra were used. The luminescence polar izat ion was measured on an instrument descr ibed by Sevchenko et al. [4] . The lifetime of the excited state was determined using the one-quantum method [5]. All the measurements were car r ied out in aqueous solution containing 0.1 m o l e / liter phosphate buffer at pH 6.5 at 20~

The electronic absorpt ion spec t rum of the NADPH: quinone oxidoreductase enzyme is given in Fig. 1. Three absorpt ion bands a re observed with maxima at 276, 405, and 454 nm. The maximum at 276 nm c o r r e - sponds to the protein segment of the molecule and has a molar extinction coefficient of 1.4 -105 liters �9 mole -1 ~ cm -1 per mole of protein. This value for the molar extinction coefficient charac te r izes proteins containing a large number of a romat ic amino acid res idues , which has been shown for other flavoproteins [6]. The nature of the band at 405 nm is discussed below. The FAD component in the enzyme gives r i se to the absorption max- imum at 454 nm with a molar extinction coefficient of 5 �9 103 l i t e r s , mole -1 �9 cm-l~

Upon excitation of the long-wavelength band of the enzyme, typical flavin luminescence with a maximum at 530 nm is observed, and the luminescence spec t rum is given in Fig. 2. The luminescence band of the en- zyme pract ical ly coincides with the luminescence band of FAD. The luminescence permits a more thorough study of the enzyme proper t ies using the cor rec ted luminescence excitation spec t rum. The cor rec ted lumi- nescence excitation spectrum obtained at awavelength of 550 nm (Fig. 2) has maxima at 370 and 450 nm, which are also charac te r i s t i c of FAD. However, in the luminescence excitation spec t rum of the enzyme, a relat ive change in the intensities of the 370- and 450-nm bands is observed relat ive to the FAD excitation spect rum. The coincidence of the luminescence spec t ra of the enzyme and of FAD, as well as of the frequencies of maxima

Transla ted f rom Zhurnal Prikladnoi Spektroskopii, Vol~ 24, No. 5, pp~ 834-838, May, 1976. Original ar t ic le submitted March 27, 1975.

This material is protected by copyright registered in the name of Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. No part [ of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, I microfilming, recording or otherwise, without written permission o f the publisher. A copy of this article is available from the publisher for $7.50. ]

591

g .lo -5 1,5

8.1g-, J mole-1 �9 cm-1. liters

o,5

280 J20 ,76O ~gO ~0 q80A, nm

i,, rel. units

,YgO qgg 500 6gO R, nm

Fig. 1 Fig. 2 Fig. 1. Absorption spec t rum of the NADPH: quinone oxidoreduetase enzyme.

Fig. 2. Daminescence (a), luminescence excitation (b), and the c o r r e - sponding luminescence polarizat ion spec t ra (c and d) of the NADPH: quinone oxidoreductase enzyme; e) difference luminescence excitation spec t rum.

in their excitation spec t ra , indicates that the isoalloxazine ring of the flavin which accounts for its lumines- cence does not part icipate in binding with the protein matr ix and is present in an aqueous environment.

Proof that the FAD molecule is bound to the protein may be found in the observat ion of luminescence polar izat ion of an aqueous solution of the enzyme. In th i s case, the FAD molecule loses severa l of its ro t a - t ional degrees of f reedom upon binding to the high-molecular-weight b iopolymer . The luminescence po la r iza - t ion spec t rum of the enzyme is given in Fig. 2c. The degree of polar izat ion is 0.12. This value is smal l r e - lative to the limiting degree of luminescence polar izat ion of FAD in glycer in, which is equal to 0.44. The attachment of the FAD molecule with the protein apparently is not rigid, which leads to part ial rotational de- polar izat ion of the FAD luminescence.

The conditions for the dissociat ion of the enzyme complex were studied in order to clar i fy the nature of the binding of the FAD molecule to the protein. Denaturation of the enzyme by t r ich loroace t ic acid or by boil- ing leads to the precipi tat ion of the protein, but not to the precipitat ion of the flavin. A change in the confor- mation of the enzyme leads to dissocia t ion of the flavin molecule f rom the protein. Fur the rmore , separat ion of the FAD molecule takes place upon prolonged dialysis of the enzyme (on the order of 100 h). This precludes a covalent bond in the complex studied. Li terature data [7] indicate that in flav odoxins, the flavins are attached to the prote in through phosphate groups by ionic bonds which depend on the conformational state of the protein.

The use of r iboflavin as a standard [8] permitted us to determine the luminescence quantum yields of FAD and of the enzyme (4.1 and 1.5%, respect ively) . The inclusion of the flavin molecule into the enzyme leads to quenching of the luminescence, as well as to a dec rease i n the lifetime of the excited state f rom 5.6 nsec for FAD to 2.2 nsec for the enzyme. The observed changes in the quantum yield and the lifetime of the excited state are of the same order of magnitude, which might indicate luminescence quenching by coll ision [9] through nonradiative excitation energy t r ans fe r f rom the FAD molecule to the surrounding molecules . The reduction in the lifetime of the excited state may lead to an increase in the luminescence polarization.

In order to determine the effect of collisions of FAD molecules with protein to which it is not attached and the ions in solution on the quantum yield and the luminescence polarization, aqueous solutions of FAD (concentration of 10 -5 mole / l i te r ) with proteins which do not bind flavins {trypsin and papain) were studied. The F A D - - p r o t e i n concentrat ion ra t io was 1 : 1 . The observed luminescence of FAD was depolarized and the quantum yield was unchanged. Thus, at the enzyme concentrat ion employed, luminescence quenching of flavin does not take place on account of collisions of FAD molecules with proteins to which they are not attached and the observat ion of luminescence polar izat ion of the enzyme indeed indicates a specific mechanism for the in- t e rac t ion of the flavin and the prote in component of the enzyme. This hypothesis is further supported by the observat ion that boiling for 2 min, which leads to damage of the protein s t ruc ture and cleavage of the flavin, causes complete depolarizat ion of the FAD luminescence.

Thus, the following model for the spatial a r rangement of the components of the active center of the NADPH: quinone oxidoreductase enzyme may be proposed on the basis of the resul ts obtained. The isoallox- azine ring of the FAD molecule which is responsible for the luminescence and for the hydrogen t rans fe r r e a c - t ion in the course of the redox reac t ion is not found on the surface of the protein but ra ther within some sor t of

592

J J

J J J

J

Fig. 3. Possible a r rangement of the FAD mole- cule in the enzyme.

fold and does not part icipate in binding the FAD molecule to the protein. This ring is surrounded by amino acid res idues and is available to the external medium. In this case, the isoalloxazine ring is bound to the FAD molecular skeleton only by the N 9 - C single bond and is capable of being displaced relat ive to the skeleton and, thus, relat ive to the protein matrix, which leads to part ial depolar izat ion of the flavin luminescence. During this motion, the isoalloxazine r ing undergoes coll is ion with the surrounding amino acid res idues of the protein or with their solvation shells and with ions adsorbed near the active center of the enzyme, which leads, in the final analysis , to the quenching of the FAD luminescence within the enzyme. This scheme for the a r rangement of the FAD molecule in the active center of the enzyme (Fig. 3) was formulated taking into account the confor- mation of FAD in solution [10]o

As shown above, con t ra ry to the identical luminescence spec t ra of the enzyme of the FAD, a red is t r ibu- t ion of band intensities in their excitation spec t ra is observed. Normalizing the excitation spec t ra according to the long-waYelength band, we car r ied out the subtract ion (Ienz-- IFAD)530nm over the entire luminescence excitation band. The difference spec t rum is given in Fig. 2, which features two luminescence excitation bands with maxima at 350 and 410 rim.

Upon excitation at the 350-nm band, luminescence with a maximum at 435 nm was observed. A possible source of this luminescence may be the reduced form of pyridine nucleotide (NADH2) which has s imi la r lumi- nescence charac te r i s t i cs when bound to protein [11]. The observed luminescence is polarized and the degree of polar izat ion is 0.25, which supports the hypothesis of the binding of NADH 2 to the protein. Indeed, boiling the enzyme for 2 min leads to a shift in the luminescence band maximum to 445 nm (with excitation at 350 nm), a, v e l l a s t o adrop in thevalue for the luminescence polar izat ion in this band to 0.05, which is charac te r i s t i c for free NADH 2 [9]. Thus, these resul ts indicate that the enzyme contains the reduced form of pyridine nucleo- t ide. This was not unexpected as NADH 2 part icipates in the enzyme-cata lyzed react ion as a hydrogen atom donor. The procedure for isolating the enzyme apparently does not cause any dest ruct ion of the e n z y m e - - sub- s t ra te complex.

The maximum obtained in the difference excitation spec t rum at 410 nm (Fig. 2) is also observed in the enzyme absorpt ion spec t rum (Fig. 1). Upon excitation at 410 nm, we were unable to find a specific lumines- cence on the background of the ftavin luminescence. On the basis of the resul ts of Lyamin and Sirota [12], we may propose that this maximum corresponds to the half-reduced form of the molecule FAD + , which, s imi lar to FAD, may be present as an enzyme component. However, this proposal requires further detailed examina- t ion.

The luminescence properties of the protein segment of the enzyme are similar to the properties of other proteins [13] and thus we did not examine these properties carefully. The maximum in the absorption spec- trum lies at 340 nm. The protein luminescence is polarized and the degree of polarization is 0.27 upon exci-

tation at 300 nm.

593

G

"?.2

0 7

~ - ~

-8

220 260 J00 A~nm I ,

is, Fig. 4. Circular d ich ro i sm spec t rum of the NADPH: quinone oxidoreductase enzyme. The mean molec- u la r weight of an amino acid res idue was taken to be 110 [14].

The c i r cu la r d ichro i sm method was used to study the conformational features of the prote in segment of the molecule. The c i rcu la r d ichro i sm spec t rum is given in Fig. 4, Three absorpt ion bands of differing in- tens i t ies a re observed. The bands with maxima at 209 and 222 nm belong to the a - s t r u c t u r e of the prote in [14]o The third band at 273 nm belongs at the adenine res idue of the FAD and NADH 2 molecules [15]. The contribution of these components to the molecular e l l ipt ic i ty of the prote in does not exceed 2% in the 200-250- nm band. This permi ts us to calculate the percentage composit ion of a - s p i r a l s , /~-structures, and unpolarized tangles i n t h e enzyme molecule studied by the model s t ruc tu re [16] and i sod ichro ic methods [17]. The calcu- lations by these methods give s imi la r resu l t s and indicate that the enzyme has 10 �9 1% s - s p i r a l s , about 40% .J -s t ruc tures , and the r e s t of the pro te in (50%) consists of an unpolarized tangle. Unfortunately, these methods do not permi t us to calculate the lat ter two values with an accuracy g r ea t e r than ~10%.

We express our grat i tude to G. G. Petrovski i and ~. P. Ti tovets for ass i s tance in isolating the enzyme.

L I T E R A T U R E C I T E D

1. M. Dixon, Biochim. Biophys. Acta, 226, No. 2, 259 (1971)o #

2. E . P . Ti tovets and Go G. Petrovski i , in: Current Problems in Neuropathology and Neurosurgery [in Russian], Minsk (1973), p. 138.

3. E . P . Ti tovets , E. F. Lunets, and G. G. Petrovski i , in: Abstracts of the Scientific Reports to the Third All-Union Biochemical Meeting [in Russian], Vol. 1 (1974), p. 71.

4. A . N . Sevchenko, M. A~ Katibnikov, and Yu. V. Star tsev, Vestn. Be lorus . Gos. Univ., Ser . 1, 1, No. 1, 32 (1970).

5. I . I . Gulakov, Author 's Abst rac t of Candidate 's Disser ta t ion , Minsk (1973). 6. J. Ao D'Anna and G. Tollin, Biochemis t ry , 11, No. 6, 1073 (1972)~ 7. M . L . Macknight and J~ M. Gillard, Biochemis t ry , 12, No~ 21, 4200 (1973). 8o G. Weber and Fo Wo J. Teal , Trans~ Faraday S .c . , 53, No~ 5, 646 (1957). 9. A . M . Sarzhevskii and Ao No Sevchenko, Absorpt ion Anisotropy and Molecular Light Emiss ion [in Rus-

sian], Minsk (1971). 10. R . H . Sarma, P. Dannies ,and No O. Kapian, Biochemis t ry , 7, No. 12, 4359 (1968). 11. A. D~ Wirier, W.B. Novoa, and G. W. Scherwert , J~ Amero Chem. S .c . , 79, No. 24, 6571 (1957). 12. Go N~ Lyamin and V. G. Sirota, in: Advances in Photonics [in Russian], No. 3 (1973), p. 63. 13. E . A . Chernitskii , Luminescence and the Structural Lability of Proteins in Solution and in the Cell [in

Russian], Minsk (1972). 14. I. A~ Bolotina, in: Molecular Biology, INT Series [in Russian], Vol. l , I zd , Vsesoyuz. Inst. Nauch. Tekh.

Inform. (1973), p. 61. 15. D . W . Miles and Do W. Urry , Biochemis t ry , 7, No. 8, 2791 (1968). 16. N~ Greenfield and G. D. Fasman, Biochemis t ry , 8, No. 22, 4108 (1969)~ 17. Y . P . Myer, Biophys. J . , 9, No. 6, A215 (1969).

594