THEO CHEM Journal of Molecular Structure (Theochem) 364 (1996) 139-149 Semiempirical and ab initio study of closed and open shell derivatives of IO-methylisoalloxazine: a model of flavin redox states Michael Meyera’*, Holger Hartwigb, Dietmar Schomburg” aGBF (Gesellschaft ftir Biotechnologische Forschung), Abt. Molekulare Strukturforschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany bInstitut fiir Physikalische Chemie, Abt. Chemische Physik. Universitiit Kiel, Olshausenstr. 40. D-24098 Kiel, Germany Received 6 November 1995; accepted 16 January 1996 Abstract The structures of a series of neutral and charged derivatives of IO-methylisoalloxazine in three different redox states have been optimized with the semiempirical PM3 method and with ab initio methods at Hartree-Fock level. The isoalloxazine ring is planar in the oxidized and one electron reduced state, except for the cations with a proton at Nl, and it is folded in the two electron reduced state. Single point MP2 and self consistent reaction field calculations have been used for the analysis of the energies in the gas phase and in a polar medium. The energy difference between the cations with a proton at Nl and 02 is small for each redox state and depends on the solvent. Protonation and deprotonation energies have been calculated. Keywords: Ab initio calculation; Flavin; lo-Methylisoalloxazine; Redox reaction; Salvation; Tautomer PM3 1. Introduction The biochemically important structures ribo- flavin, flavin mononucleotide (FMN) and flavin ade- nine dinucleotide (FAD) contain the isoalloxazine nucleus shown in Fig. 1. The diversity of reactions of these enzyme co-factors is enormous because the oxidized quinone-type flavins (substituted isoalloxa- zines) can be involved in both one- and two-electron reductions, leading to the flavosemiquinone and fla- vohydroquinone. The mentioned species are ampho- teric, i.e. they can exist as anions, neutral molecules or cations, and the structure can be folded about the N5-NlO axis depending on the redox state. * Corresponding author. It has been proposed that the degree of the fold may modulate the reactivity of the flavin redox system [l]. Thus it is interesting to compare the fold of isoalloxazine derivatives with the FAD of flavoenzymes. In continuation of our work on the flavoenzyme glucose oxidase [2] and on small model molecules [3,4] we selected lo-methylisoal- loxazine as a model to determine the properties of the flavin redox system. We preferred to use the methyl substituted species because the bulkier methyl group should resemble the biologically important molecules such as riboflavin more clo- sely than the system with hydrogen at NlO. Furthermore, much experimental work on free fla- vins [5], which we use for comparison, has been done with alkyl substituted species to prevent a 0166-1280/96/%15.00 0 1996 Elsevier Science B.V. All rights reserved PIZ SO166-1280(96)04491-O
Transcript
THEO CHEM
Journal of Molecular Structure (Theochem) 364 (1996) 139-149
Semiempirical and ab initio study of closed and open shell derivatives of IO-methylisoalloxazine: a model of flavin redox states
Michael Meyera’*, Holger Hartwigb, Dietmar Schomburg”
Received 6 November 1995; accepted 16 January 1996
Abstract
The structures of a series of neutral and charged derivatives of IO-methylisoalloxazine in three different redox states have been optimized with the semiempirical PM3 method and with ab initio methods at Hartree-Fock level. The isoalloxazine ring is planar in the oxidized and one electron reduced state, except for the cations with a proton at Nl, and it is folded in the two electron reduced state. Single point MP2 and self consistent reaction field calculations have been used for the analysis of the energies in the gas phase and in a polar medium. The energy difference between the cations with a proton at Nl and 02 is small for each redox state and depends on the solvent. Protonation and deprotonation energies have been calculated.
Keywords: Ab initio calculation; Flavin; lo-Methylisoalloxazine; Redox reaction; Salvation; Tautomer PM3
1. Introduction
The biochemically important structures ribo- flavin, flavin mononucleotide (FMN) and flavin ade- nine dinucleotide (FAD) contain the isoalloxazine nucleus shown in Fig. 1. The diversity of reactions of these enzyme co-factors is enormous because the oxidized quinone-type flavins (substituted isoalloxa- zines) can be involved in both one- and two-electron reductions, leading to the flavosemiquinone and fla- vohydroquinone. The mentioned species are ampho- teric, i.e. they can exist as anions, neutral molecules or cations, and the structure can be folded about the N5-NlO axis depending on the redox state.
* Corresponding author.
It has been proposed that the degree of the fold may modulate the reactivity of the flavin redox system [l]. Thus it is interesting to compare the fold of isoalloxazine derivatives with the FAD of flavoenzymes. In continuation of our work on the flavoenzyme glucose oxidase [2] and on small model molecules [3,4] we selected lo-methylisoal- loxazine as a model to determine the properties of the flavin redox system. We preferred to use the methyl substituted species because the bulkier methyl group should resemble the biologically important molecules such as riboflavin more clo- sely than the system with hydrogen at NlO. Furthermore, much experimental work on free fla- vins [5], which we use for comparison, has been done with alkyl substituted species to prevent a
0166-1280/96/%15.00 0 1996 Elsevier Science B.V. All rights reserved PIZ SO166-1280(96)04491-O
140 M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149
0
0 Fig. 1. Numbering scheme of the isoalloxazine ring.
tautomerization of isoalloxazine to alloxazine with hydrogen at Nl instead of NlO.
Hall and colleagues used the MIND0/3 method to optimize the structures of oxidized and reduced flavins [6] and ab initio calculations of closed and open shell species have been reported by Platen- kamp and co-workers at fixed solid state structures or standard geometries [7-g]. We now extend the previous studies to tautomers, which have not been studied before. The structures were optimized com- pletely at the semiempi~cal PM3 level to determine the most stable species and to test the accuracy of the method, because computational efficiency enables this method to be used for the investigation of model systems for the active site of enzymes. The most relevant species were then investigated by ab initio methods. We present the first nonempirical study with complete structure optimizations to obtain information about the dependence of the fold of the isoalloxazine ring on the redox state and on the protonation of the nitrogen Nl, which leads to a steric interference with the methyl group at NlO. The calculations allow a meaningful com- parison of the fold angle to be made, because the results are not overlayed by the influence of different substituents of the ring system. In addition we compare the protonation energies and discuss redox equilibria. We expect our energies to be more accurate than those of previous studies, because they do not depend on non- optimized structures. Finally, self consistent reaction field calculations have been carried out to estimate the dependence of the energies on a polar medium.
2. Methods
Semiempirical calculations have been carried out with the PM3 method [lo] as implemented for restricted (RHF) and unrestricted Hartree-Fock (UHF) calculations in MOPAC 6.0 [1 11. The structures have been optimized using eigenvector following Ref. [12] starting from various planar and non- planar geometries. Subsequently the minima were characterized by a diagonalization of the force- constant matrices.
Ab initio calculations have been carried out using the direct SCF method of the programs GAMES (131 and GAUSSIAN 92 [14]. The radicals have been treated with the restricted open shell Hartree-Fock (ROHF) method; for the other molecules the RHF method was used. All geo- metries have been optimized completely employing the 6-3lG* basis set [15]. Single point calculations at the HF/6-3 1 G* geometries have been carried out with the 6-3lG** basis [16] and at the MP2 level. For the structure optimizations with ab initio methods we used initial bond lengths and angles derived from the solid state structure of the oxi- dized lo-methylisoalloxazine [17] or from the semiempirical calculations. Non-planar “butterfly”- type structures with a fold along the N5-NlO axis were used as initial points for structure optimizations based on the eigenvector following method. The self consistent reaction field (SCRF) method [18] based on the Onsager model has been used to compute the energy changes due to non- specific electrostatic interactions with a polar medium.
3. Results
In the following section we present the results of our calculations on heats of formation with semi- empirical methods and ab initio calculations of the energies for three redox states derived from lo- methylisoalloxazine. By analogy to the abbrevia- tions for the flavin redox system [5], we refer to the neutral oxidized lo-methylisoalloxazine as MI,,H, to the deprotonated anion as MI, and to the protonated cation as MI,,H2f. For the discussion of specific tautomers we give the
M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149 141
MLH (0% MI,,H,+ (NlN3)
~,rIw4)
Fig. 2. Neutral tautomers of the oxidized IO-methylisoalloxazine.
protonated nitrogen or oxygen atoms in brackets, e.g. M&H (N3) shown in Fig. 2. The numbering scheme of the isoalloxazine ring is shown in Fig. 1. The abbreviations for the neutral one- and two- electron reduced species are MI’H2 and MI,&Hs.
3.1. Semiempirical heats of formation
The heats of formation AH, were calculated with PM3 for all neutral and charged species of each redox state. A selection of the results for the most stable tautomers is listed in Table 1. MI,,H (N3) (Fig. 2) has the lowest heat of formation (AHf = -12.7 kcal mol-i) of the neutral oxidized tautomers. The heats of formation of the tautomers with hydrogen at oxygen MI,,H (02) (AHf = - 1.2 kcal mol-‘) and MI,,H (04) (AHf = -0.6 kcal mol-‘) are somewhat higher. The heat of
W I
MIoxH; (02N3)
0 Fig. 3. Protonated and deprotonated derivatives of the oxidized lo-methylisoalloxazine.
formation of the anion MI, is -45.1 kcal mol-‘; for the cations depicted in Fig. 3, the method pre- dicts that AH, of the tautomer MI,,Hz (02N3) (138.0 kcal mol-‘) will be lower than that of MI,,H,f (NlN3) (141.2 kcal mol-‘). PM3 predicts that AH, of a further species MI,,H$ (N304) (149.7 kcal mol-‘) will be higher than that of MI,,H; (NlN3).
The semiquinone type molecules resulting from a one electron reduction are shown in Figs. 4 and 5. AH, = -26.4 kcal mol-’ was determined for the neutral radical MI’H2 (N3N5). For MI’H2 (NlN3) we get a slightly less negative result of -24.3 kcal mol-‘. MI’H- (N3) with AHf = -76.9 kcal mol-’ is the most stable radical anion. MI’H; (02N3N5) (AHf = 116.3 kcal mol-‘) and MI-H: (NlN3N5) (AHf = 118.9 kcal mol-‘) are the most stable radical cations.
142
Table 1
hf. Meyer et aLlJournal of Molecular Structure (Theochem) 364 (1996) 139-149
Heats of formation AH, (kcal mol-‘) of IO-methylisoalloxazine derivatives
Derivative Value Derivative Value Derivative Value
ML _ -45.1
MI’H- (02) -64.0
(N3) -76.9
(04) -66.3
MIdHi (NlN5) -13.0
(02N5) -77.0
(N3N5) -88.0
(02) -1.2
(N3) -12.7
(04) -0.6
(NlN3) -24.3
(N104) -14.3
(02N3) -19.1
(0204) -17.9
(02N5) -14.4
(N304) -18.8
(N3N5) -26.4
(N102N5) -24.6
(NlN3N5) -39.4
(N104N5) -24.4
(02N3N5) -33.2
(0204N5) -28.6
(N304N5) -29.3
ML& (NlN3) 141.2
(02N3) 138.0
(0204) 148.7
(N304) 149.7
(N3N5) 151.6
MI’H; (NlN304) 130.4
(NlN3N5) 118.9
(N104N5) 129.8
(02N304) 129.1
(02N3N5) 116.3
MI,,& (NlN304N5) 116.9
(NlN3N5N5) 110.7
(02N304N5) 115.7
(02N3NSN5) 109.0
A selection of two electron reduced hydroqui- nones is shown in Fig. 6. MI,&Hs (NlN3N5) and MIrdH; (N3N5) are the most stable neutral and anionic species in the two-electron reduced state. MIrdHl (02N3N5N5) (AHr = 109.0 kcal mol-‘)
yH3
Fig. 4. Neutral and tautomers of the one electron reduced lo-
methylisoallazine.
and MIredHz (NlN3N5N5) (AHr = 110.9 kcal mol-‘) are the most stable reduced cations. A dou- ble protonation of Nl or N3 instead of N5 leads to much higher heats of formation.
3.2. Ab initio energies
The total energies of the reference species are listed in Table 2 and relative energies of the corre- sponding tautomers are given in Table 3. The diketone form of neutral MI,,H with hydrogen bound to N3 is energetically favoured relative to the tautomers with hydrogen at 02 or 04. At RHF/6-31G* the energy differences are 16.4 kcal mol-’ and 20.5 kcal mol-‘, respectively. The cationic species MI,,Hl (NlN3) is 3.2 kcal mol-’ less stable than MI,,H,f (02N3), whereas the energy of the tautomer MI,,Hl (N304) is 11.7 kcal mol-’ higher than that of MI,,H,f (NlN3).
The ROHF energy of the neutral species MPH2 (N3N5) is lower than that of the tautomer MI’H;, (NlN5). For the radical cation MI’H: (NlN3N5) the energy is 1.4 kcal mol-’ higher at ROHF/ 6-31G* than the energy of M1.H: (02N3N5).
For the reduced state two cations with one hydrogen bound to N3 and two hydrogens at N5
M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149 143
CH, I
MI% (N3)
Ml%,+ (NlN3N5)
(02N3N5)
0
Fig. 5. Protonated and deprotonated derivatives of the one elec- tron reduced IO-methylisoalloxazine.
have been investigated (Fig. 6). The energies of these species, differing only in the position of the fourth hydrogen, are similar. At the RHF/6-31G* level the energy of MIrdH,f (NlN3N5N5) is 0.4 kcalmol-’ below thatofMIrdHz (02N3N5N5).
Additional polarization functions on hydrogen lower the energies of all species with a proton on oxygen to a larger extent than the energy of the corresponding species with a proton on nitrogen. The order of the relative energies is not altered except for the cations in the reduced state. The single point MP2 corrections to the energy lead to a strong decrease of the energy of the cations with a proton at Nl relative to the ones with a proton at 02.
The protonation and deprotonation energies of the neutral species are listed in Table 4. The proto- nation energy is defined as the difference between
0
MI&H, (NlN3N5)
CH,
CH3
I Ii
MIdH,+ (NlN3NSNq
7H3
MI,,H,+ (02N3N5N5)
Fig. 6. Neutral and charged tautomers of the two electron reduced IO-methylisoalloxazine.
the total energy of the protonated cation and the corresponding neutral species. The deprotonation energy is the difference between the energy of the neutral species and the anion.
4. Discussion
4.1. Heats of formation and energies
Three of the tautomers of oxidized neutral MI,,H are shown in Fig. 2. According to the
144 M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149
semiempirical and ab initio calculations MI,,H (N3) is more stable than the tautomers with hydro- gen bound to oxygen. So far these tautomers have not been detected by spectroscopic experiments. For the neutral radical species MI’H;! (N3N5) the heat of formation is lower than that of MPH2 (NlN3), which is also in agreement with the current knowledge about flavin chemistry [5]. The lowest heats of formation of the cations in all redox states refer to species with hydrogen at 02 such as
M&H,’ (02N3). For the corresponding tautomers with hydrogen at Nl (e.g. MI,,Ht (NlN3)) we obtained a slightly higher result. A comparison of the heats of formation of the cations in different redox states shows that the difference between the 02 and Nl protonated species decreases from 3.2 kcal mol-’ for the quinones to 1.9 kcal mol-’ for the hydroquinones. An intermediate value of 2.6 kcal mol-’ is found for the semiquinones.
The relative energies from the ab initio
Table 3 Relative energies (kcal mol-‘)
PM3 HF/6-3lG* MP2,‘6-3lG*//HF/6-3lG* SCRF
M&H (N3) 02 11.5 04 12.1
16.4 14.7 16.4 14.4 20.5 18.8 19.1 19.6
MI,H; (NIN3) 02N3 -3.2 04N3 8.5
-3.2 -5.0 -1.9 -0.7 11.7 9.9 9.6 13.3
MI’H2 (N3N5) NlN3 2.1 6.6 6.7 8.3
MI’H: (NlN3N5) 02N3NS -2.6 -1.4 -1.3 1.2
M&H; (N3N5) N3N5a 5.4 1.8 1.6 2.6 2.4
M&H; (NlN3NSNS) 02N3N5N5 -1.7 0.4 -1.1 1.3 4.6
’ Planar.
M. Meyer et al/Journal of Molecular Structure (Theochem) 364 (1996) 139-149
Table 4 Protonation and deprotonation energies (kcal mol-‘)
145
HF/6-31G*
Protonation of MI,,H (N3) MLH: (NlN3) 233.3
(02N3) 236.5 (N304) 221.6
Protonation of MI’H2 (N3N5) MI’H; (NlN3N5) 241.1
(02N3N5) 242.5
Protonation of MI,,H, (NlN3N5) MhH: (NlN3N5N5) 232.7
(02N3N5NS) 232.1
Deprotonation of MI,,H (N3) MI, 365.8
Deprotonation of MI’H2 (N3N5) MI’H- (N3) 341.1
Deprotonation of MIxdH, (NlN3N5) MImiH; (N3N5) 342.5
calculations resemble the relative heats of forma- tion for the cations. At the HF/6-31G* level the cations for the oxidized and one electron reduced state with hydrogen at 02 were found to be energetically favoured by a small amount relative to the ions with hydrogen at Nl. For the reduced state the energy difference between the cations is the smallest one and the sign depends on the choice of the basis set. The MP2 contribution and the effect of a polar medium lead both to a stronger decrease of the energies of the cations with a proton at Nl relative to the ones with a proton at 02. Though the energy differences are quite small, MI,,Ht (02N3) seems to be the most stable catio- nit oxidized species in the gas phase. Single point calculations at HF/6-3 1 lG**//HF/6-3 lG* lead to an energy difference of -4.5 kcal mol-’ relative to MI,,Hi (NlN3), which is close to the HF/6- 31G**//HF/6_31G* result in Table 3. The zero point vibration does not influence the relative energy noticably, because the energy contribution is almost identical for both species. The zero point energies are 0.21364 H and 0.21367 H for MI,,Hz (NlN3) and MI,,H,f (02N3), respectively.
The protonation energy of the flavosemiquinone
MI’Hz (N3N5) is higher than the corresponding energy for the oxidized state MI,,H (N3) and for the two electron reduced state MIrdH, (NlN3N5). Thus a protonation should occur most easily in the one electron reduced state yielding MI’H; (02N3N5) or MI’H; (NlN3N5). The energy being required for a deprotonation of the neutral species yielding an anion is highest for the oxidized state (see Table 4).
Furthermore, the energies of Table 2 give some hints concerning the mechanism of reduction of MI,,H (N3). An electron transfer in the first step yielding MI’H- (N3) leads only to a slight reduc- tion of the energy of 17.9 kcal mol-’ at HF/6- 31G*. A proton transfer leading to (NlN3) gives much lower energies in a and may be followed by the electron which again reduces the energies.
4.2. Solvation efects
ML& first step transfer,
The SCRF method based on the Onsager model has been employed to estimate the solvent influence on the relative energies of the tautomers. The solute is placed in a spherical cavity embedded in a
146 M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149
continuous medium with a die!ectric E = 80. Cavity radii between 4.64 and 4.99 A have been derived from the 0.001 a.u. density adding 0.5 A to account for the nearest approach of solvent molecules [19].
The relative energies in Table 3 show that a polar solvent stabilizes the cations with a proton at Nl to a larger extent than the tautomers with a proton at 02, which is similar to the MP2 contributions to the energies. MI’HZ (NlN3N5) and MIredHl (NlN3N5N5) are more stable than the correspond- ing species with a proton at 02 in a polar medium, which is in agreement with current opinions in flavin chemistry [5]. In contrast to experimental results the computations predict that MI,,H,’ (NlN3) will be less stable than MI,,H,f (02N3). However, in solution the protonation takes place at Nl instead of 02 in the oxidized state [20] and the X-ray structure of IO-methylisoalloxazine hydro- bromide dihydrate [21] clearly shows a hydrogen at Nl, which forms a hydrogen bond with the oxy- gen of a water molecule. In our calculations neither the direct interaction with water nor a bromide anion is included. Thus the results are not directly comparable. However, the single point MP2 calcu- lation leads to a decrease of the relative energy of 1.3 kcal mol-’ and the SCRF calculation gives a diminution of 2.5 kcal mol-’ . Therefore SCRF cal- culations at the correlated level are likely to show that MI,,Hz (NlN3) is the most stable oxidized cation in a polar medium. Furthermore, the appli- cation of a spherical cavity is only a crude approx- imation of the molecular shape, leaving empty space above and below the molecular planes. The solvent effects are probably somewhat underesti- mated, because increasing the cavity radii by 1 A causes a smaller decrease of the relative energy between MI,,Hz (NlN3) and MI,,H{ (02N3) (1.2 kcal mol-’ instead of 2.5 kcal mol- ).
An interesting aspect of the flavin system is the possibility of redox reactions in different ionic states, depending on the pH of the solution. The following equilibria may occur:
MI,,H (N3) + MIredH3 (NlN3N5)
= 2 MI’Hz (N3N5) (1)
MI, + MIredH; (N3N5)
= 2 MI’H- (N3) (2)
Table 5
Differences between the total energies (kcal mol-‘) of reactants
The total energies of Table 2 can be used to pre- dict whether a comproportionation or dispropor- tionation is energetically favoured. Using the RHF energies of the oxidized and two-electron reduced species with lowest energy in each redox state and the ROHF energies for the radicals, we predict that reaction (1) will lead to a disproportionation, because the sum of the energies of the species on the left side is lower than twice the energy of the radical on the right side. For the equilibria of the ionic species we predict a comproportionation. These calculations are in agreement with experi- ments because it has been found that in neutral aqueous solution disproportionation occurs, whereas at low pH the semiquinone is favoured. The anionic flavosemiquinone may be obtained in water-free dimethylformamide with t-butoxide as a base [5]. The energy differences are listed in Table 5. Their magnitude is drastically reduced in a polar medium relative to the gas phase. The energy dif- ference between products and reactants for the anionic state is particularly small because the energy of MI& is very much lower in a polar medium.
4.3. Molecular structures
Selected structural parameters of the neutral tau- tomers MI,,H (N3), MI’H2 (N3N5) and MIredH3 (NlN3N5) from ab initio calculations are given in Table 6. A comparison of the data for the different redox states shows that the bond lengths and angles of the benzene ring of the isoalloxazine system are hardly affected by reduction. Major differences between MI,,H (N3) and MI’H2 (N3N5) are the shortened bonds C4-C4a and C4a-ClOa of the
M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149 141
Table 6
Structural parameters of neutral oxidized and one- and two-electron reduced derivatives of IO-methylisoalloxazine (distances in .&,
Fold angles (degrees) of non-planar derivatives of IO-methyl-
radical. The bond C4a-N5 of course is elongated if N5 is protonated. Similarly the additional proto- nation of Nl in MIredH, (NlN3N5) results in a
isoalloxazine
MI,,H; (NlN3) 4.1
MI’H2 (NlN3) 8.0
MI’H; (N 1 N3N5) 7.1
MI,,H; (N3N5) 26.3
MIredH, (NlN3N5) 27.4
MI,..,H; (NlN3N5N5) 23.5
MI,,,,H; (02N3NSN5) 11.4
much longer Nl-ClOa distance compared to the other species. Furthermore, C2-N3 is somewhat shorter and C4a-N5 is longer than the corre- sponding distances in the radical. The most inter- esting changes involve a deviation from planarity by folding along the axis N5-NlO. This fold may be described by the angle of the least squares plane of the atoms Nl, C2, N3, C4, C4a, ClOa, N5, NlO
148 M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149
and the plane C5a, C6, C7, C8, C9. C9a, N5 and NlO. We calculated this angle similarly to the method of Blow [22]. The fold angles for the non- planar species are listed in Table 7. Our structure optimizations for oxidized species converged to structures which are almost or exactly planar. For MI,,H (N3) and its deprotonated anion we obtained a planar structure with the in-plane hydrogen of the methyl group directed to Nl. The planarity is in close agreement with an X-ray study [17]. We derived a small fold angle of 2.7” from the solid state coordinates of MI,,H (N3). The protonation of Nl disturbs the planarity of the ring somewhat. For MI,,H: (NlN3) we calcu- lated 4.1”, which agrees well with the solid state angle of 4.2” [21]. The structures of the radicals are similarly planar; only a hydrogen at Nl leads to a major deviation of the isoalloxazine ring from planarity. The fold angles for MI’H2 (NlN3) and MI’H: (NlN3N5) are 8.0” and 7.1”, respectively. Previous estimates of the fold are conflicting. Evidence for the planarity of flavosemiquinones has been provided by electron nuclear double resonance (ENDOR) [5], whereas non-planar structures have been proposed for the unsub- stituted isoalloxazine radicals on the basis of UHF calculations for different assumed geometries [9]. The species in the reduced state show the strongest deviations from planarity. We calculated 23.5” for MI,,dH: (NlN3N5N5), 27.4” for MIredH3 (NlN3NS) and 26.3” for MIredHy (N3N5). A hydrogen at 02 instead of Nl leads to a considerably smaller fold angle of 11.4” for MIredHz (02N3N5N5). A bulkier substituent at Nl or C9 seems to cause a slight increase of the fold angle. For example an angle of 31” has been found in the X-ray structure of 9-bromo-1,3,7,8,10- pentamethyl- 1,5-dihydroisoalloxazine [23] whereas 27.4” has been calculated for the corresponding neutral species MIredH3 (NlN3N5). The energy difference between the planar structure and the minimum structure of MIredHi (NlN3N5) increases from 1.8 kcal mol-’ at RHF/6-31G* to 2.4 kcal mol-’ upon inclusion of electron corre- lation. Similarly, the barrier is increased in a polar medium.
The fold angle of the oxidized FAD in an enzyme can deviate substantially from planarity. Hecht
et al. pointed out that the isoalloxazine ring of glu- cose oxidase [2] is already folded in the oxidized state. The fold angle of the co-factor is 16.1”, which almost approaches fold angles of the reduced state species of free MI. This structure is fixed by hydrogen bonds and steric interactions in the protein. A fold of similar magnitude has been found by Vrielink et al. in the crystal structure of cholesterol oxidase [24], also being a member of homologous proteins called GMC oxidoreductases [25]. In contrast to the fold of the co-factor of these enzymes an angle of 3.1” appears in glutathione reductase [26]. This structure corresponds to the oxidized or one electron reduced derivatives of MI.
5. Conclusions
We have presented structure optimizations for derivatives of lo-methylisoalloxazine. The ring system is folded along the N5-NlO axis in the two-electron reduced state. Planar structures exist in the oxidized and one-electron reduced state, except for a small fold of the cations with a proton at Nl (Table 7). Relative energies of tautomers from PM3 and ab initio calculations are in qualitative agreement. We found that the energies of the cations with a proton at Nl and 02 are close to each other and depend on the medium. SCRF theory can predict the relative order of the energies of cationic tautomers for the one- and two-electron reduced states; for the oxidized state a higher level of theory is necessary. The cation with a proton at 02 is the most stable cation in the oxidized state in the gas phase (E = l), whereas the cations with a proton at Nl are most stable for each redox state in a polar medium (E = 80). The small energy differences suggest that not only N2 but also 02 may be involved enzymatic in proton transfer reactions. The deprotonation energy of MI,,H (N3) is much reduced in a polar medium.
Acknowledgement
We thank H.J. Hecht for critical discussions.
M. Meyer et al./Journal of Molecular Structure (Theochem) 364 (1996) 139-149 149
References
[l] C.S.J. Walpole and R. Wrigglesworth, Oxido-reductases- Flavoenzymes, in M.I. Page and A. Williams (Eds.), Enzyme Mechanisms, Royal Society of Chemistry, London, 1987, p. 507.
[2] H.J. Hecht, H.M. Kalisz, J. Hendle, R.D. Schmidt and D. Schomburg, J. Mol. Biol., 229 (1993) 153.
[3] M. Meyer, J. Mol. Struct., 273 (1992) 99. [4] M. Meyer, J. Mol. Struct. (Theochem), 304 (1994) 45. [5] F. Mtiller, Free flavins: syntheses, chemical and physical
properties, in F. Miiller (Ed.), Chemistry and Biochemistry of Flavoenzymes, Vol. 1, CRC Press, Boca Raton, 1991, p. 1.
