ELSEVIER THEO CHEM Journal of Molecular Structure (Theochem) 417 (1997) 163-168 Density functional study of isoalloxazine and C4a-hydroperoxidihydroisoalloxazine Michael Meyer* Received I9 December 1996: accepted 7 February 1997 Abstract The structures of isoalloxazine and C4a-hydroperoxidihydroisoalloxazine have been computed at B3LYP63lG* level. Solvent effects on relative energies of tautomers and protonation equilibria have been investigated using the SCI-PCM model. The results have been discussed in relation to biochemical properties of flavin coenzymes. 0 I997 Elsevier Science B.V. Kqwmds: Density functional theory; Flavin; Isoalloxazine; Solvent model: Redox reaction 1. Introduction Isoalloxazine 2 is a heterocycle with an important role in flavoproteins for biochemical redox reactions [ 1,2]. Flavins, substituted isoalloxazines, can be involved both in one and two electron reductions leading to flavosemiquinones (Fig. 1, middle) and flavohydroquinones (Fig. 1, bottom), which can be protonated or deprotonated depending on the pH. The oxidation of 7 can be achieved with molecular oxygen (Fig. 2). This density functional study describes the influence of a medium on isoalloxazine derivatives. In addition to the radicals 4-6, hydroperoxides being potentially involved in the oxidative half reaction * Present address: Biocomputing, Institut ftlr Molekulare Biotechnologie, Postfach 100 8 13, D-07708 Jena, Germany. Tel.: +49-364-1656203: fax: +49-364-1656210: e-mail: [email protected]have been investigated. The structures of these instable species, which are not available from the experiment, may yield information about interactions with other residues when superimposed with the cor- responding coenzyme at the flavoprotein active site. In addition to the commonly accepted species 3,6 and 9 with a proton at N I tautomeric cations 14. 15 and 16 with a proton at 0201 have been investigated (Fig. 3). According to semiempirical and ab initio calculations [3], the energies of these species are close to the ones of N 1 protonated ions and the recently a tautomer of 8 with a hydrogen at 0201 has been proposed as an intermediate of an enzymatic redox reaction 141. Furthermore, some seeming discrepancies between previous theoretical and experimental studies have been investigated. Previous ab initio calculations predicted that the radicals 4-6 are folded about the N5-NIO axis (5.61, whereas EPR-studies have indi- cated a planar structure of substituted radical cations 171. Similarly, different estimates exist for the energy 0166- 1280/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PI/ SO I66- 1280(97)00064-X
Transcript
ELSEVIER
THEO CHEM
Journal of Molecular Structure (Theochem) 417 (1997) 163-168
Density functional study of isoalloxazine and C4a-hydroperoxidihydroisoalloxazine
Michael Meyer*
Received I9 December 1996: accepted 7 February 1997
Abstract
The structures of isoalloxazine and C4a-hydroperoxidihydroisoalloxazine have been computed at B3LYP63lG* level.
Solvent effects on relative energies of tautomers and protonation equilibria have been investigated using the SCI-PCM model. The results have been discussed in relation to biochemical properties of flavin coenzymes. 0 I997 Elsevier Science B.V.
Kqwmds: Density functional theory; Flavin; Isoalloxazine; Solvent model: Redox reaction
1. Introduction
Isoalloxazine 2 is a heterocycle with an important
role in flavoproteins for biochemical redox reactions
[ 1,2]. Flavins, substituted isoalloxazines, can be involved both in one and two electron reductions leading to flavosemiquinones (Fig. 1, middle) and flavohydroquinones (Fig. 1, bottom), which can be protonated or deprotonated depending on the pH. The oxidation of 7 can be achieved with molecular
oxygen (Fig. 2). This density functional study describes the
influence of a medium on isoalloxazine derivatives. In addition to the radicals 4-6, hydroperoxides being potentially involved in the oxidative half reaction
have been investigated. The structures of these instable species, which are not available from the experiment, may yield information about interactions
with other residues when superimposed with the cor- responding coenzyme at the flavoprotein active site. In addition to the commonly accepted species 3,6 and 9 with a proton at N I tautomeric cations 14. 15 and 16 with a proton at 0201 have been investigated (Fig. 3). According to semiempirical and ab initio calculations [3], the energies of these species are close to the ones of N 1 protonated ions and the recently a tautomer of 8 with a hydrogen at 0201 has been proposed as an intermediate of an enzymatic redox reaction 141. Furthermore, some seeming discrepancies between previous theoretical and experimental studies have been investigated. Previous ab initio calculations
predicted that the radicals 4-6 are folded about the N5-NIO axis (5.61, whereas EPR-studies have indi- cated a planar structure of substituted radical cations 171. Similarly, different estimates exist for the energy
0166- 1280/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved
PI/ SO I66- 1280(97)00064-X
164 M. Meyer/Journal of Molecular Structure (Throchem) 417 (1997) 163-168
H’ Ii’
0 361.6 132.1 329.4 269.1 317.8 283.1 _
10 0
6.7
‘1 7 / ; N O Y scr, N
N ‘H
0 3
H’ 240.9 276.5 288.7
2.3
H* 7.27.9 267.3 280.9
co
9
Fig. I. Redox states of isoalloxazine with protonation and deprotonation energies (kcal/mol) for 6 = I ,4 and 78.5 with flavin pK, values [I]. The
oxidised state is shown in the top row, the one-electron reduced state in the middle and the two-electron reduced state in the bottom.
7 + 0,
/ i NYo OTf NH N
H i,? ‘...I 1,
Fig. 2. Potential steps of the oxidative half reaction. Intramolecular
hydrogen bonds are indicated.
difference between the planar and the folded con- formation of I ,5-dihydroisoalloxazine 8. Ab initio
calculations yielded a high energy difference of 30 kcal mol-’ [6], whereas semiempirical calculations of reduced flavins predicted a low barrier to planarity of 2 kcal mol-’ [8].
2. Experimental
The calculations have been carried out with GAUSSIAN 94 [9] using the B3LYP/6-3 lG(d) hybrid density functional method [ IO,1 11. Single point calcu- lations with the self consistent isodensity method (XI-PCM) implemented in GAUSSIAN 94 have been performed to take into account an unspecific influence of a medium with a dielectric constant E = 4 for a protein [12] and E = 78.5 for water.
Table I 9 relative to the corresponding 02a protonated Total energies (H) cations.
B3LY P SCI-PCM“ SCI-PCMh
1 -753.59092
2 -754.16710
3 -753.53699
4 -7S-1.22676
5 -754.76673
6 -755.15012
7 -753.83194
7‘ -7S4.83068
x -7ss.27200
8’ -7SS.370.56
9 --755.73421
9‘ -755.73391
10 not stable
11 -90.5. I60 I7
12 -905.71297
13 -7S4.18773
14 -153.s39s4
15 -755.15167
16 -7ss.73400
-753.65752 -753.68432
-754.18246 -754.19082
-753.61210 -754.64196
-754.28171 -754.30279
-754.78176 -754.79002
-7ss.22337 -7SS.2SOlO
-794.Xx635 -754.90640
-754.884.59 -754.9044s
-755.38469 -755.39172
-755.38490 -755.39309
-7SS.81062 -755.84073
-7SS.XO968 -755.83955
-9OS.21390 -90.5.23361
-905.72x03 -905.73643
-754.19837 -7.54.20404
-7S4.6065.5 -754.63193
-755.21671 -755.240s 1 -755.80334 -755.82961
” 6 =-I.
h c = 7x.5
k Planar.
3. Results
The total energies of the investigated molecules are
summarized in Table I, the protonation and deproto- nation energies are shown in Fig. 1. According to the relative energies in Table 2, alloxazine 13 is always more stable than the tautomer isoalloxazine 2. The energies of the cations 14 and 15 with a proton at
02a are lower than the ones of 3 and 6 in the gas phase, whereas the opposite holds in a medium, which leads to a signiticant stabilization of 3. 6 and
Tat+ 2
Relative energie\ of tautomer?, and conformers (kcal mol.‘)
B3LYP SCI-PCM” SCI-PCM’
13-2 -13.0 -10.0 -8.3
14-3 -1.6 5.6 6.3
15-h -I .o 3.5 6.0
16-9 0. I 4.6 7.0
T-7 0.x I.1 I.2
V-8 0.9 -0.1 -0.9
9'-9 0.2 0.6 0.7
‘le =4.
h 6 =7x.5.
L Planar.
The neutral and ionic molecules l-6 are planar, the
two electron reduced species (flavohydroquinones) 7. $9 are folded 14.7, 12.0 and 2 1.4” about the NS-N 10 axis.
4. Discussion
Experimental structures of flavin radicals related to 4-6 have not been reported. Previous ab initio calcu- lations have indicated that the radicals 4-6 are folded
about the N5-N IO axis [5,61, whereas a more planar structure is in agreement with EPR spectra of flavin cation radicals corresponding to 6 [7]. The present structure optimizations show that 4-6 are indeed planar like the oxidized ones 1-3, whereas the two electron reduced species are folded. The gas phase
energy differences 0.8,0.9 and 0.2 kcal molK’ between the planar and the folded structures of 7, 8 and 9 are
particularly small. The energy difference between the planar and folded structure of 1,5-dihydroisoalloxa- zine 8 is similar to the one obtained with MINDO/.? [S] for two electron reduced fiavins. but it is much smaller than the 30 kcal mol-’ determined previously with ab initio calculations [7], which had to be carried out at fixed assumed geometries and without using
polarization functions since the computer resources were much smaller at the time.
The more bulky methyl substituent of IO-methyl isoalloxazine enhances a fold of the ring system. The oxidized IO-methylisoalloxazinium cation corre- sponding to 3 has a small fold angle of 3.1’ and the radical cation is folded about 8.0” 1.11. Similarly. the two electron reduced species are folded two a larger extent. The energy difference between the planar and the folded structure of 7 is increased to values between 1.6 and 2.6 kcal mol. ’ upon methyl substitu- tion, depending on the method of computation.
The properties of isoalloxazine derivatives depend on the environment. The source of some discrepancies between previous calculations and experimental data
is the neglect of solvent effects. In a gas phase the cations 14 an 15 with a proton at 0201 have a lower energy than tautomers 3 and 6 with a proton at N I. This deviation from experimental results 1 I I is an effect of the medium and the application of the
SCI-PCM model interchanges the order. Similarly, it has been concluded from NMR-studies, that the fold angle of flavohydroquinones is much lower than the computed ones as a consequence of the solvent [ 13 1. This interpretation agrees with the present calcula-
tions. According to Table 2, the energy of the folded conformation 8 is 0.9 kcal mall’ lower than the one of the planar structure. In water the energy difference is
reversed to -0.9 kcal mol-’ and in a protein environ- ment the energy difference is particularly small. The
relative energies of the ions 7 and 9 are not reversed. The isoalloxazine ring in the oxidized state is folded
by hydrogen bonds in some flavoproteins, for example the angle is 16. lo in glucose oxidase [ 141 and a similar
angle has been found in cholesterol oxidase [ 1.51 and trimethylamine dehydrogenase ] 161, whereas it is
almost planar in other cases, e.g., glutathione reduc- tase [ 171 and the old yellow enzyme [ 181. It has been proposed that flavoproteins become a more powerful reducing agent by bending the isoalloxazine ring [ 191. This requires that the energy rises with the fold angle in the oxidized state since the energy difference between planar and non-planar conformations in the two electron reduced state are small. Only limited conclusions can be drawn from the fold angle of the flavin on the preferred enzymatic mechanism. A strong fold might be a hint for a two electron reduc- tion of a suitable substrate. Such an interpretation is in
agreement with the proposed mechanisms of choles- terol 1201 and glucose oxidase 12 l] having folded FAD coenzymes. For these enzymes a hydride trans- fer (2 - 7) (in a single or in multiple steps) has been discussed for substrate oxidation. Furthermore, a fast initial two-electron transfer to FMN and a formation of FMNHz (2 - 8) has been proposed for trimethyl- amine dehydrogenase, which is followed by a slow one electron transfer from FMNH? to an Fe-S cluster [22]. But an almost planar structure might result both from one and two electron reductions so that no con- clusions can be derived. It is also possible that the activation energies are modified to a higher degree by the fold.
The protonation and deprotonation energies listed in Fig. 1 depend drastically on a medium. For example, the protonation energy of 2 rises from 232.1 in vacuum to 269.0 in protein environment and 283.1 kcal mall’ in water. The deprotonation energy decreases from 361.5 to 329.4 in a protein
and 317.8 kcal mol-’ in water. In other words, more energy is required to form the anion 1 from the neutral
molecule 2 and less energy is required for the forma- tion of 2 from the cation 3 in a low dielectric medium compared to water. This holds also for the other redox
states. A medium decreases in general the difference in protonation and deprotonation energies for the
different redox states. For a hydride transfer to isoalloxazine (2 - 7) the
energy of the system decreases 4 16.6 in the gas phase, 441.7 in a protein and 449.0 kcal mol-’ in water.
The qualitative agreement between energy calcula- tions and spectroscopic measurements indicate that the SCI-PCM model is more suitable for the investi- gation of solvent effects for large planar systems than
the Onsager model [23], which has been used to study model systems for biochemical problems [3,24]. The
disadvantage of the Onsager model for planar mole- cules, a fixed spherical cavity for the solute molecule, has been replaced in the SCI-PCM model by an iso- density surface taking the coupling between electron density and cavity into account. Previous calculations based on the Onsager model at Hartree-Fock level have led to drastically smaller solvent effects on
protonation and deprotonation energies for 1 O-methyl isoalloxazine [3]. In Fig. I, the pK, of flavins [I J is given in addition to the (de)protonation energies.
Since the applied quantum mechanical solvent model does not take into account all contributions to the free energy of solvation, no estimation of the pK,
has been attempted. From kinetic investigations, it has been concluded
that 7 forms a radical pair with 02 reacting to 10 by spin conversion [25]. 12 is not stable in water and decomposes in 2 and hydrogen peroxide.
For a triplet state of 10. no local energy minimum
with a typical carbon oxygen bond was determinable in vacuum. From all starting structures, a dissociation
of the C4a-00 bond occurred and O2 moved away. It seems likely that a protein environment may stabilize
a triplet adduct because the oxidation in a protein can be orders of magnitude faster than the oxidation of free flavins in solution [25]. Optimizations for the singlet state of 10 lead to an instantaneous tauto- merisation yielding 11, which indicates that N5-H is more acidic than 00-H in 10 and 11.
Selected structural parameters of species involved in the oxidative half reaction are listed in Table 3. One
I67
Table 3
Selected bond lengths CA) and angles (“1
I1 12
r(C4ao) I.522 1.469 ? r(O0)
r(OH)
rtH...NS)
r(H...OZa)
Q(C IOaC‘Iao)
<(C4aOO I Q(OOH)
7(C I OaC‘hOO I K4aOOH 1
I.456
0.9u.c
1.91-l _
100.x 106.6
9x.4
167.9 -2x.4
I.446 0.08 I
2.179
103.7
108.2
101.9
167.5
72.0
0
might suspect that the tetrahedral C4a causes a non- planar structure, but similarly to the experimental
structures of model substances [26] 11 and 12 are almost planar, only C4a is located somewhat outside of the plane formed by the other atoms of the ring system. In 11, an intramolecular hydrogen bond NS...H-00 with a distance of 1.914 A is formed whereas in 12 a somewhat longer bond OO- H...02a exists in the energetically most favourable conformation.
In addition to the commonly accepted reaction of reduced flavins with oxygen at C4a in biochemical
relevant systems ] I]. C IOa-hydoperoxidihydroiso- alloxin derivatives have been proposed 127). For
such an isomer of 12 no energy minimum exists. This may be a consequence of the fact that in the experimental work N I -substituted flavins have been used. but not for the investigated model systems and
for the biochemically relevant coenzymes.
OH
5. Conclusions
Density functional calculations at B3LYP/6-3 lG* level have shown that isoalloxazine is planar in the oxidized and one electron reduced state, whereas it is folded in the two electron reduced state. The barrier to
planarity is small and the fold angle depends on a substituent at NIO. A medium can modulate the degree of the 1,5-dihydroisoalloxazine 8 fold. C4a- hydroperoxidihydroisoalloxazine 12, a potential inter- mediate of the oxidative flavin half reaction, has an almost planar ring system and an intramolecular
16
Fig. 3. Alloxazlnc and tautomeric cations In different redox \t;ltes.
I68 M. Me~er/Jouvml of Moleculur Structure (Theochm) 417 (1997) 163-I68
hydrogen bond exists in the lowest energy conforma- tion. For the corresponding ClOa-isomer no local energy minimum exists.
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