34

Biochimica et Biophysica Acta, 526 (1978) 34--41 © Elsevier/North-Holland Biomedical Press

BBA 68507

HYDROGEN BONDING OF FLAVOPROTEIN I. EFFECT OF HYDROGEN BONDING ON ELECTRONIC SPECTRA OF FLAVOPROTEIN

K I C H I S U K E N I S H I M O T O a, Y O S H I T A K A W A T A N A B E a and K U N I O Y A G I b

aDepartment of Chemistry, Faculty of Science, Osaka City University, Osaka 558, and blnstitute of Biochemistry, Faculty of Medicine, University of Nagoya, Nagoya 466 (Japan) (Received D e c e m b e r 28th, 1977)

Summary The effect of hydrogen bonding on the transition energy and the oscillator strength of the isoalloxazine nucleus of flavins was studied by the molecular orbital method. Among the possible hydrogen bondings examined, characteristic spectral shifts were found for the hydrogen bondings at N(1) and N(5) of the nucleus. The hydrogen bonding at N(1) resulted in the shift of the first absorption band towards blue and that of the second one towards red. On the other hand, the hydrogen bonding at N(5) resulted in the shifts of both the first and the second band towards red. The spectral characteristics reported on Clostridium MP and Desulfovibrio vulgaris flavodoxin coincided with the calculated results. The application of the calculated results to Iyamino acid oxidase (Iyamino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) led to the conclusion that hydrogen bonding occurs at O(12), N(3)H, O(14) and N(5) of the isoalloxazine nucleus. The occurrence of hydrogen bondings at O{12), N(3)H, and O(14) is favorable for N(5) of the isoalloxazine nucleus to accept electron from an electron donor.

Introduction Upon complex formation of riboflavin with phenol, the first absorption band of the isoalloxazine shifted towards red, and the interaction energy of riboflavin • phenol complex was 6.1 Kcal/mol. The interaction was ascribed to the hydrogen bonding between the isoalloxazine nucleus of riboflavin and hydroxyl group of phenol [1]. Therefore, the shift of the first absorption band of the isoalloxazine towards red observed in the reconstruction of flavoproteins Abbreviation:

VSIP, valence state ionization potential.

35 such as the old yellow enzyme [2] and l>amino acid oxidase (Iramino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) [3] is considered to be ascribed to the occurrence of hydrogen bonding between the isoalloxazine nucleus of the coenzyme and the apoenzyme. Theorell and Nygaard [4] proposed the occurrence of hydrogen bonding in the complex formation between the coenzyme, riboflavin 5'-phosphate, and the apoenzyme of the old yellow enzyme. Later, Kotaki et al. [5] studied the effect of hydrogen bonding occurring on the isoalloxazine on its absorption spectrum by using a model system. These results led us to make a theoretical approach to the relation between the occurrence of hydrogen bonding on possible groups of the isoalloxazine and its absorption spectrum. The present paper deals with the results obtained along this line in order to gain a better understanding of the significance of hydrogen bonding in flavoproteins. Methods Molecular orbital calculation. In Fig. 1, possible hydrogen bondings at the hereto atoms (nitrogen and oxygen) of the isoalloxazine are shown. In order to examine the effect of such hydrogen bondings on the electronic spectra of the isoalloxazine, we carried out self-consistent field molecular orbital calculation including configuration interaction based on ~-electron approximation, using both variable/3 approximation [6,7] for a two-center core.integral and the Nishimoto-Mataga formula [8] for a two-center electron repulsion integral. The values of valence state ionization potential (VSIP) were approximately estimated in a way similar to those reported previously [9]. Hydrogen bonding can be expressed as a resonance hybrid of the following structure:

X-H---Y ~ (X-H)---Y+** X----(H-Y)÷ Therefore, the hydrogen bond formathon has an effect of decreasing the value of VSIP of electronegative atom X and an effect of increasing that of electronegative atom Y. Accordingly, upon formation of hydrogen bonding, the values of VSIP of carbonyl oxygen and of pyridine type nitrogen (=N-) should increase, while that of pyrrole type nitrogen (-NH-) should decrease. To check the effects of the different values of VSIP on the transition energy of the isoalloxazine nucleus, we varied the value of pyridine type nitrogen, N(1) or N(5), for a rather wide range, viz. from 14.12 eV (no hydrogen bond) to 17.12 eV. The results are shown in Fig. 2. From this figure, one can expect that the difference in the value does not change the characteristics in spectral shifts, though the magnitude of the shifts differed. Since the main purpose of this study is to clarify the characteristic change in the electronic spectra and electronic structure of flavin upon formation of hydrogen bondings, the VSIP values of the hetero atoms of the isoalloxazine without hydrogen bondings [7] were modified by adding or subtracting an arbitrary value, 1.0 eV. The values thus obtained are summarized in Table I. Another series of values was also obtained by adding or subtracting 0.5 eV. The excitation energies were calculated by the method of configuration interaction. All singly-excited configurations were allowed to interact.

36

I 9

I

~2

I

r oyN O

I

I

N/~-.~NH

,

0

I

NO HYDROGENBONDING

.T~

i

I I

~

0

II

I

I , .F NN'-rO"

i

0

0 t I

N.T

V. O(12),N(3)H,O(14)HYDROGEN BONDING

yO

. ~

0

I I

VIII.

HYDROGENBONDING

I i

N(1),O(lZ),N(3)H,O(14)-

IX N(1),O(12),N(5)HYDROGENBONDING

i i

I

0 I I

I I

X N(1),O(12),O(14),N(5)

HYDROGENBONDING

Xl

0 I I

I I

NyN rO

T ~ N .~N ~ 0 / ~ ] ' - ~ N~ , , ~ NH..

~'K~N-~NH

I

~'~../K'-N~NH , 0

HYDROGENBONDING (Cl. MP FLAVODOXIN)

~"--~N"-F~N'-~ O"

N(1),O(IZ)HYDROGEN BONDING

N.rO

"--

I I

N(1),0(12),0(14)-

VI

N..~m-.~ O"J

0

i I I

0

I l l . N(5)-HYDROGENBONDING

~N-~N'~O '''/

IV. N(1),N(5)HYDROGEN BONDING

vii.

i I

N(1)-HYDROGEN BONDING

N-~N-.~ 0

i i

I

0

.

"

O(12),N(3)H,O(14),N(5)

HYDROGENBONDING (D vulgaris FLAVODOXIN)

.-"

-.

XII. N(1),O(12),N(3)H,O(14), N(5)-HYDROGENBONDING

Fig. 1. Possible s t r u c t u r e s of h y d r o g e n - b o n d e d i s o a l l o x a z i n e . TABLE I P A R A M E T E R S (VSIP) FOR T H E M O L E C U L A R O R B I T A L C A L C U L A T I O N OF H Y D R O G E N BONDED I S O A L L O X A Z I N E VSIP v a l u e s w e r e i n d i c a t e d in eV. N u m b e r i n g o f a t o m s o f t h e i s o a l l o x a z i n e is i l l u s t r a t e d in Fig. 1, T y p e L T h e s e p a r a m e t e r s w e r e e s t i m a t e d b y c o n s i d e r i n g a r e s o n a n c e h y b r i d o f h y d r o g e n b o n d i n g as d e s c r i b e d in text. Type of hydrogen bonding

Atom N(1)

N(3)

N(5)

N(10)

O(12)

O(14)

C

I II III IV V VI VII VIII IX X XI XII

14.12 15.12 14.12 15.12 14.12 15.12 15.12 15.12 15.12 15.12 14.12 15.12

26.70 26.70 26.70 26.70 25.70 26.70 26.70 25.70 26.70 26.70 25.70 25.70

14.12 14.12 15.12 15.12 14.12 14.12 14.12 14.12 15.12 15.12 15.12 15.12

26.70 26.70 26.70 26.70 26.70 26.70 26.70 26.70 26.70 26.70 26.70 26.70

17,70 17.70 17,70 17,70 18,70 18.70 18,70 -18.70 18.70 18.70 18.70 18.70

17.70 17.70 17.70 17.70 18.70 17.70 18.70 18.70 17.70 16.70 18.70 18.70

11.16 11.16 11.16 11.16 11.16 11.16 11.16 11.16 11.16 11.16 11.16 11.16

37

N(1) VSIP (eV) 14.12~

HYDROGENBONDINGAT N(1)

---m

15"12F

ILI)F 300

N(5)

VSIP (eV)

iiiii ., 300

m

50 400 WAVELENGTH(rim)

450

HYDROGENBONDINGAT N(5)

, 350 400 WAVELENGTH(nm)

450

Fig. 2. D e p e n d e n c e of the w a v e l e n g t h of a b s o r p t i o n h a n d s o n the v a l u e s of VSIP u p o n f o r m a t i o n of h y d r o gen b o n d i n g a t N(1) or N(5). The c a l c u l a t e d values of t h e first a n d t h e s e c o n d a b s o r p t i o n b a n d in wavel e n g t h were p l o t t e d a g a i n s t t h e values of VSIP. U p p e r figure, for the case of h y d r o g e n b o n d i n g a t N ( 1 ) ; l o w e r figure, for t h e case of h y d r o g e n b o n d i n g a t N(5). I, t h e first a b s o r p t i o n b a n d ; II, t h e s e c o n d absorption band.

Results

Calculated electronic spectra. Fig. 3 shows the calculated values of the transition energy and the oscillator strength (intensity of absorption) of the first and the second absorption band. The visible absorption bands obtained from the calculation without hydrogen bonding interactions locate at 414 and 332 nm. They are far shorter than those of the observed wavelengths, 445 and 350 nm. However, this disagreement is not important in considering the effect of hydrogen bonding interaction on the spectral characteristics [9]. As can be seen from the figure, upon hydrogen bonding at N(1) of the isoalloxazine the first absorption band shifts towards blue, while the second one does not shift (compare I with II in Fig. 3). Upon hydrogen bonding at N(5) of the isoalloxazine both absorption bands shift towards red (compare I with III in Fig. 3). Since the calculation was made by using VSIP values obtained by adding or subtracting an arbitrary value, 1.0 eV (see Methods), another calculation was made by using the values obtained by adding or subtracting 0.5 eV. The result showed the same characteristic shifts of the absorption bands except that the changes in transition energy were approximately half of the above data. Comparison of the data shown in Fig. 3 with each other indicates that the additional occurrence of hydrogen bondings at O(12), N(3)H and O(14) gives a minute effect on the shifts of the absorption bands caused by hydrogen bondings at

38

WAVELENGTH (nm) 360 380 400

340 I

I

I

l

I

t

ill

i

IV

3.00

I(o.5551 2.98

1(o.514/ 3.14 (0.544) 3.13 (0.538) 3.13 (0.537) 3.02

, (0.I19)

VII I

Vlll

3.68 (O.131) 3.67

j (0.159)

3.55 ,(O.lll) 3.50 ,(0.123) 3.50 (0.133) '3.50 (0.149)

IX

X XI XII

3'.8

I

I

3.6

I

(0.509) I

3~72

VI

440 I

1(o.5o8/

3.67 (0.132)

V~

I

3.13 (0.545) 2.86

3.58 (0.094) 3.58 (0.I09)

Ill

420 I

2.99

3.74 ,(0.083/ 3.74 ,(0.115)

I

I

J

I

3.4

I(o.5541 3.00

1(o.55o) 2.85 (o.51511 ,

i

3'. 2

3.00 J(0.547) 3.0

2.8

TRANSITIONENERGY(eV) Fig. 3. Calculated values for the transition energy and the oscillator strength o f various t y p e s of h y d r o g e n b o n d e d i s o a U o x a z i n e . T h e values o f the t r a n s i t i o n e n e r g y for various t y p e s o f h y d r o g e n - b o n d e d isoa l l o x a z i n e s h o w n in Fig. 1 w e r e i n d i c a t e d b y vertical bars b o t h in e V and in w a v e l e n g t h scale. T h e h e i g h t o f the bars s h o w s the relative i n t e n s i t y o f the relevant a b s o r p t i o n band. T h e n u m e r i c a l values in the figure i n d i c a t e the t r a n s i t i o n e n e r g y and the o s c i l l a t o r s t r e n g t h (in Parenthesis).

N(1) and/or N(5). Accordingly, when hydrogen bondings occur at all of the possible hetero atoms of the isoalloxazine, the shifts of both absorption bands (from I to XII in Fig. 3) are similar to those from I to IV in Fig. 3. In the former case, however, the red shift of the second absorption band is remarkable. The changes in the oscillator strength upon formation of hydrogen bonding(s) also showed a characteristic behavior. The hydrogen bonding at N(1) increases the oscillator strength of both absorption bands remarkably (compare I with II in Fig. 3), and that at N(5) slightly increases the oscillator strength of the both (compare I with III in Fig. 3). When hydrogen bondings occur at O(121, N(3)H and O{141, change in oscillator strength of the first absorption band is minute, while that of the second one is remarkable (compare I with V in Fig. 3). When hydrogen bondings occur at all of the possible hetero atoms, oscillator strength of both the absorption bands increases remarkably (compare I with XII in Fig. 3). Electron acceptability. It is also important to examine the influence of hydrogen bondings on the redox potential of the isoalloxazine. The redox potentials are related to the energy of the lowest unoccupied molecular orbital

39 -3.6

-3.75 ~ -3.83 \

-3.8

\ -3.94

-3.93 -3.92 -3.99

-4.0

-4.00

-4.02

._J

\ \-4,11

-4.13

ii i

-4.2

~

-4.19

I

II

Ill

IV

V

Vl

VII

VIII

IX

X

-4.19

XI

XII

Fig. 4. E n e r g y levels o f the l o w e s t u n o c c u p i e d m o l e c u l a r orbital ( L U M O ) o f various t y p e s of h y d r o g e n b o n d e d i s o a l l o x a z i n e . T h e n u m e r i c a l v a l u e s in the f i g u r e i n d i c a t e the l o w e s t u n o c c u p i e d m o l e c u l a r orbital e n e r g y in eV.

[10,11]. As shown in Fig. 4, the hydrogen bonding interactions shown in Fig. 1 all enhance the electron affinity of the isoalloxazine. Namely, upon hydrogen bonding interactions, flavin could receive an electron from an electron donor more easily. Among the complexes studied, the effect is marked in the cases of type IX • XII complexes. The electron acceptability of the particular atom in the isoalloxazine nucleus can be measured by the square of the atomic orbital coefficients in the lowest unoccupied molecular orbital (frontier electron density) [12]. Frontier electron density of non-hydrogen bonded isoalloxazine is essentially similar to the reported result [13,14]. Namely, without hydrogen bonding, N(5) of the nucleus is the most dominant atom to accept an electron. The change of the frontier electron density at N(5) upon formation of hydrogen bondings is shown in Table II. Hydrogen bondings at O(12), N(3)H and O(14) remarkably increase the frontier electron density at N(5). The frontier electron density at N(5) decreases due to the hydrogen bonding at N(5). However, this is restored in part by the formation of the additional hydrogen bondings at O(12), N(3)H and O(14).

T A B L E II THE FRONTIER

ELECTRON

DENSITY AT N(5)

T h e frontier e l e c t r o n d e n s i t y w a s c a l c u l a t e d f r o m t h e a t o m i c orbital c o e f f i c i e n t a t N ( 5 ) i n t h e l o w e s t u n o c c u p i e d m o l e c u l a r o r b i t a l , as r e p o r t e d b y F u k u i et al. [ 1 2 ] . No hydrogen bonding Hydrogen bonding at N(5) Hydrogen bondings at O(12), N(3)H and O(14) H y d r o g e n b o n d i n g s a t O ( 1 2 ) , N ( 3 ) H O ( 1 4 ) and N ( 5 )

0.236 0.223 0.242 0.227

40

Discussion The present molecular orbital calculation indicates that the occurrence of hydrogen bonding at N(1) or N(5) markedly modifies the absorption spectrum of the isoalloxazine and that the hydrogen bondings at the other atoms of the isoalloxazine give little influence on its absorption spectrum. To check the validity of these results, flavodoxins are the best instances, since the occurrence of hydrogen bondings on the isoalloxazine nucleus was reported by X-ray crystallographic analysis. In the flavodoxin crystal obtained from Clostridium MP, hydrogen bondings occur as represented in Fig. 1, VIII [15,16]. Another flavodoxin obtained from Desulfovibrio vulgaris was reported to possess hydrogen bondings as shown in Fig. 1, XI [17,18]. The results of the molecular orbital calculation in the relevant cases were illustrated in Fig. 3. Although the conformation of flavodoxins in solution may not be the same with that in crystals, the theoretical data reported in this paper are in good accord with the data reported by Mayhew and Ludwig [ 19]. From the above argument, it seems reasonable to correlate the characteristic change in the absorption spectrum of the isoalloxazine nucleus with the occurrence of hydrogen bondings. On the basis of this assignment, D-amino acid oxidase was examined for the hydrogen bondings involved in the formation of the complex of FAD and the apoenzyme. The absorption spectrum of FAD in aqueous media possesses the peaks of the first and the second absorption band at 450 and 375 nm, respectively. Upon complex formation with D-amino acid oxidase apoenzyme, the shift towards red with a slight decrease in absorbance is found in the first absorption band, while appreciable shift is not found in the second absorption band [3]. This spectral change accords with that from Fig. 3, XII -XI obtained theoretically. This implies that the hydrogen bonding at N(1) of FAD in aqueous solution disappears upon its complex formation with the apoenzyme, while N(5) still interacts with the apoenzyme or remains to interact with water molecule via hydrogen bonding. The hydrogen bonding at N(5) increases the electron affinity of the isoalloxazine nucleus, while it slightly decreases the magnitude of the frontier electron density at N(5). However, if additional hydrogen bondings occur at O(12), N(3)H and O(14), the electron affinity of the isoalloxazine nucleus increases and N(5) still possesses relatively high electron acceptability. This seems to be the case for both D. vulgaris flavodoxin and D-amino acid oxidase. When substrate combines with D-amino acid oxidase, amino nitrogen seems to interact with N(5) of the isoalloxazine nucleus of the coenzyme [20,21]. In this case, if the hydrogen bonding at N(5) is still remaining, the amino nitrogen should interact with N(5) in a sterically possible way. Otherwise, the hydrogen bonding at N(5) disappears prior to the interaction between N(5) and the amino nitrogen of the substrate. In the latter case, the frontier electron density of N(5) increases, which accelerates the electron flow from the substrate to the coenzyme. In any way, hydrogen bondings occurring at O(12), N(3)H and O(14) are considered to be one of the important factors for the catalytic activity of flavoproteins.

41

Acknowledgment The authors are grateful to the Data Processing Center, Kyoto University, for its generous permission to use the FACOM 230-75 computer. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17, 18 19 20 21

Yagi, K. and Matsuoka, Y. (1956) Biochem. Z. 328, 138--145 TheoreU, H. and Akeson, ~ . (1956) Arch. Biochem. Biophys. 65,439--448 Yagi, K. and Ozawa, T. (1962) Biochim. Biophys. Acta 56, 413---419 Theorell, H. and Nygaard, A. (1954) Acta Chem. Scand. 8, 1649--1658 Kotaki, A., Naoi, M. and Yagi, K. (1970) J. Biochem. 68, 287--292 Nishimoto, K. and Forster, L.S. (1965) Theor. Chim. Acta 3, 407---417 Nishimoto, K. and Forster, L.S. (1966) Theor. Chim. Acta 4, 155--165 Nishimoto, K. and Mataga, N. (1957) Z. Phys. Chem. (Frankfurt) 12, 335--338 Nishimoto, K., Nakatsukasa, K., Fujishiro, It. and Kato, S. (1969) Theor. Chim. Acta 14, 80--90 Mat~en, F.A. (1956) J. Chem. Phys. 24, 602--606 Batley, M. (1966) Thesis, University of Queensland, Australia Fukui, K., Yonezawa, T., Nagata, C. and Shingu, H. (1954) J. Chem. Phys. 22, 1423--1442 Grabe, B. (1972) Acta Chem. Scand. 26, 4084--4100 Song, P.-S., Choi, J.D., Fugate, R.D. and Yagi, K. (1976) in Flavins and Flavoproteirm (Singer, T.P., ed.), pp. 381--390, Elsevier, Amsterdam Burnett, R.M., Darling, G.D., Kendall, D.S., LeQuesne, M.E., Mayhew, S.G., Smith, W.W. and Ludwig, M.L. (1974) J. Biol. Chem. 249, 4383--4392 Ludwig, M.L., Burnett, R.M., Darling, G.D., Jordan, S.R., Kendall, D.S. and Smith, W.W. (1976) in Flavins and Flavoproteins (Singer, T.P., ed.), pp. 393---404, Elsevier, Amsterdam Watenpaugh, K.D., Sieker, L.C. and Jensen, L.H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3857--3860 Watenpaugh, K.D., Sieker, L.C. and Jensen, L.H. (1976) in Flavins and Flavoproteins (Singer, T.P., ed.), pp. 405--410, Elsevier, Amsterdam Mayhew, S.G. and Ludwig, M.L. (1975) in The Enzymes (Boyer, P.D., ed.), Vol. 12, pp. 57--118, Academic Press, New York Yagi, K. (1971) in Advances in Enzymology (Nord, F.F., ed.), Vol. 34, pp. 41--78, J o h n Wiley and Sons, New York Yagi, K., Nishikimi, M., Ohishi, N. and Takai, A. (1971) in Flavins and Flavoproteins (Kamin, H., ed.), pp. 239--260, University Park Press, Baltimore

Hydrogen bonding of flavoprotein. I. Effect of hydrogen bonding on electronic spectra of flavoprotein.

34 Biochimica et Biophysica Acta, 526 (1978) 34--41 © Elsevier/North-Holland Biomedical Press BBA 68507 HYDROGEN BONDING OF FLAVOPROTEIN I. EFFECT...
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