Chemical modification of the isoalloxazine ring of flavins and
incorporation of modified flavin derivatives into apoproteins has
proven to be a powerful tool in understanding the relationship between
protein structure and catalysis in flavoenzymes(1) . The N-10
ribityl side chain of flavins has generally been considered as only a
binding anchor with no positive role in catalysis. However, the x-ray
crystal structures of various flavoproteins such as glutathione
reductase(2, 3) , acyl-CoA
dehydrogenase(4, 5) , lipoamide
dehydrogenase(6) , and old yellow enzyme (7) showed the
involvement of ribityl hydroxyls in hydrogen bonds that could regulate
catalytically important amino acid residues at the active sites. From
the three-dimensional structure of medium chain acyl-CoA dehydrogenase,
two hydrogen bridges pointing toward the thioester carbonyl were
identified as activating the substrate(4, 5) . One of
them involves the flavin 2`-OH group and the other Glu-376. Recent
studies of Ghisla et al.(8) with
2`-deoxy-FAD-reconstituted medium chain acyl-CoA dehydrogenase
confirmed the crystal structure findings and for the first time
demonstrated a role for the ribityl side chain in catalysis. Studies
with 2`-F-arabino-FAD-reconstituted mercuric reductase (9, 39) showed that the two-electron reduced form in
which the flavin remains oxidized and the active site disulfide reduced
is destabilized by this modification and complete flavin reduction was
observed. The crystal structures of glutathione reductase and lipoamide
dehydrogenase(3, 6) have shown that the 2`-hydroxyl
group is involved in an important hydrogen bond network in the oxidized
forms of the proteins. The 2`- and 4`-hydroxyls and an adenine
phosphate oxygen atom make two intramolecular hydrogen bonds that could
play a significant role in determining the conformation of the bound
prosthetic group (Fig. 1A). In the reduced form, the
distance between 2`-OH and the active site Cys-63 decreases from 3.9 to
3.4 Å. Also Sol-601 now donates hydrogen bonds to the phosphate
oxygen and 4`-OH, so that the 2`-OH can rotate its hydrogen around to
stabilize the active site thiolate anion through a hydrogen bond,
making it possible to accommodate a buried charge generated during
catalysis (Fig. 1B). These literature reports clearly
highlight the possible importance for the 2`-hydroxyl group in
regulating protein structure and catalysis.
Figure 1:
Flavin-protein interactions
in glutathione reductase. A, protein environment of N-10 side
chain in the oxidized form of glutathione reductase (from (3) and (6) ). Hydrogen bond interactions are
represented by dotted lines. B, interaction of
2`-hydroxy group of flavin ribityl side chain with the active site
thiolate, Cys-63 in the 2-e
-reduced form of
glutathione reductase (from (2) and (3) ). The charge
transfer interaction between flavin and thiolate is represented by a wavy line. Hydrogen bond interactions are shown by dotted
lines.
Lipoamide dehydrogenase,
glutathione reductase, and mercuric reductase represent a well studied
family of flavoprotein-disulfide oxidoreductases. The catalytic
reaction mechanisms of each of them present a number of common
features(10) . The flow of electrons during catalysis in these
enzymes is to or from pyridine nucleotide to FAD to active center
disulfide to substrate disulfide. All three of these proteins exist in
three distinct redox forms, i.e. oxidized (E), two electron
reduced (EH
), and four electron reduced (EH
) states, the EH
form
being catalytically relevant. In the light of known crystal structures,
these proteins constitute an excellent set of models for
F
NMR as well as enzymatic studies with 2`-position-modified flavins. We
were interested to see how the different oxidation states of these
proteins differ structurally from one another by
F NMR
chemical shifts and also how the chemical modifications may affect
catalysis. Accordingly, we synthesized three flavin derivatives
modified at the 2`-position of the ribityl side chain i.e. 2`-deoxyriboflavin, arabinoflavin, and 2`-F-arabinoflavin.
The
fluoroflavin has the potential of being a useful
F NMR
probe. Such NMR studies employing 8-fluoroflavins provided a wealth of
information on the structure, conformation, and dynamics of
flavoproteins(11) . Of all the nuclei,
F is one of
the most effective ones because of its high abundance, extreme
sensitivity to the surrounding environment, and a large range of
chemical shifts(12, 13, 14) . Furthermore,
since the proteins normally do not contain any fluorine, one can
investigate protein-prosthetic group, prosthetic group-ligand
interactions without interference from the protein. The potential
relevance of these modified flavins is also highlighted by some recent
work with p-hydroxybenzoate hydroxylase. The flavin in this
enzyme has been shown to be mobile, being capable of moving away from
the substrate binding site under certain conditions, a phenomenon
postulated to be involved in substrate binding and
catalysis(15, 16) . The alcohol oxidase isolated from
methylotrophic yeasts has been shown to contain two different forms of
FAD(17) . NMR studies have shown that one of them as native FAD
and the other as arabino-FAD(18) . This chemical conversion of
the native FAD results from autocatalysis by the purified
enzyme(18) . Recently van Berkel et al. solved the
x-ray crystal structure for arabino-FAD-p-hydroxybenzoate
hydroxylase and showed that with p-hydroxybenzoate as
substrate, the flavin takes the ``out'' conformation, unlike
in the native enzyme, where the flavin assumes the ``in''
conformation(19) . The out conformation results in substantial
oxidase activity, producing hydrogen peroxide, instead of productive
hydroxylation.
EXPERIMENTAL PROCEDURES
Materials
Deuterium oxide and
2-fluoro-2-deoxy-1,3,5-tri-O-benzoyl-
-D-arabinofuranose
were from Sigma. D-Arabinose, 2`-deoxyribose, sodium
borohydride, 10% Pd/C, dithiothreitol, hexafluorobenzene,
4-amino-o-xylene, and barbituric acid were from Aldrich.
Preparation of Flavoproteins and Their Corresponding
Apoproteins
The holoproteins and apoproteins were prepared as described
previously: riboflavin binding protein from hen egg white(20) ;
lipoamide dehydrogenase from pig heart(21) ; glutathione
reductase from human erythrocytes(22) ; mercuric reductase from Escherichia coli W 3110 lac Iq containing the plasmid
pPS01(22) .
Apoprotein Reconstitution
Reconstitution of the apoproteins with
2`-fluoroarabinoflavin, 2`-deoxyflavin, and arabinoflavin was
accomplished by mixing an approximately 1.5-fold excess of flavin in
the appropriate coenzyme form with apoprotein and then incubating on
ice (from 30 min to overnight, depending on the protein). Excess flavin
was removed by a Centricon microconcentrator (Amicon). Unless otherwise
indicated, all samples were prepared in 0.1 M potassium
phosphate buffer, pH 7.0.
NMR Sample Preparation
Protein samples for NMR were concentrated by means of a
Centricon 30 microconcentrator to 150-200 µl and were diluted
to 400 µl with 50 µl of D
O and for the rest of the
volume with buffer.Reduced flavin and protein samples were prepared
by the addition of known excess of reducing agent to sample in the NMR
tube. The tubes were flushed with argon (space over the sample) for 15
min prior to the addition of the reductant in buffer. Argon flushing
was continued during and after the addition. The tube was then closed
with a standard plastic cap and sealed with parafilm. Color changes
associated with the reduction persisted for several days.
NMR Acquisition Parameters
Fluorine NMR spectra were recorded with a 10-mm
F probe and with a General Electric GN 500 instrument,
operating at 470 MHz. All of the NMR measurements were carried out
without proton decoupling, at 25 °C(23) . Fluorine chemical
shifts were measured by using hexafluorobenzene as an external standard
and are given in parts/million. Spectra were measured with 16,000 or
32,000 data points over spectral widths from 20 to 45 kHz. The
repetition time was 0.005 to 1 s with a 12-µs pulse (60° flip
angle).
NMR Processing
With broad lines and low signal to noise taken over a wide
spectral width, the base line of an NMR spectrum is not
flat(24, 25) . Correction routines (GEM routines IC,
FB, and BF) were used to flatten the base line. Exponential line
broadening was performed from 10 to 50 Hz.
Syntheses of Modified Flavins
The fluoroarabinoflavin was synthesized (Fig. S1) by
making use of the Tishler condensation reaction(26) .
Figure S1:
Synthesis of
2`-fluoro-2`-deoxyarabinoflavin.
2-Fluoro-2-deoxyarabinose
1 g of
2-fluoro-1,3,4,5-tetra-O-benzoylarabinose was dissolved in 40
ml of methanol + ammonium hydroxide mixture (1:1) and stirred
overnight with a magnetic stirrer. The solvents were then evaporated
with a rotary evaporator, and the obtained solid was dissolved in a
minimum volume of methanol and loaded on a (1.5
8 cm) silica
gel column. The benzoic acid and benzamide products were eluted with a
mixture of ethyl acetate and hexane (3:1). Washing the column with 100%
methanol and evaporation of the solvent gave the debenzoylated fluoro
sugar in 82% (272 mg) yield.
N-2-F-2-deoxyarabino-3,4-dimethyl
Benzimine
ortho-Xylidine (194 mg) and 245 mg of the
fluoroarabinose were dissolved in 15 ml of ethanol and refluxed for 3
h. After cooling in a refrigerator, the crystals were filtered off and
washed with cold ether to obtain 315 mg of fluoroimine in 72% yield.
Reduction of the Imine
The fluoroimine, 315 mg,
obtained in the above reaction was dissolved in 25 ml of ethanol, and
excess sodium borohydride (400 mg) was added. After stirring the
reaction mixture overnight, the excess reagent was destroyed with the
careful addition of water, the solvent was evaporated, and the solid
was diazotized without purification.
Diazotization
185 mg of freshly distilled aniline
was dissolved in 2 ml of acetic acid, and the solution was diluted with
2 ml of water. Then, 1 ml of concentrated HCl was added dropwise with
cooling. To this solution, 170 mg of solid NaNO
was added
while maintaining the temperature at less than 5 °C. The so-formed
diazonium chloride was kept at 0 °C for at least 5 min for its
complete formation. At this stage, the solid obtained in the preceding
reaction was suspended in a minimum volume of acetic acid and cooled to
0 °C. The precooled azo dye was added dropwise to the acetic acid
solution of amine, and the temperature was kept at 0 °C for 10 min.
A concentrated solution of sodium hydroxide (approximately 400 mg in 1
ml water) was added and care was taken that the pH remained below 4
(
3-4) after the addition. The reaction mixture was kept at 0
°C for 2 h. The reaction mixture was then extracted with 100 ml of
ether, and the ether layer was separated. The aqueous solution was
reextracted twice with 50 ml each of ether, and the combined organic
fractions were washed with saturated sodium bicarbonate to neutralize
acid and washed with water followed by drying over anhydrous sodium
sulfate. Evaporation of the ether gave 186 mg of azo dye (45% yield
from the imine).
2`-F-2`-deoxyarabinoflavin
185 mg of the azo dye
and 70 mg of barbituric acid were suspended in 10 ml of n-butyl alcohol. To this mixture, 2.5 ml of acetic acid was
added and refluxed for 3 h. Thin-layer chromatography on silica gel
(developed in ethyl acetate) at this stage showed no azo dye, and a
yellow fluorescent spot was observed at the base. Development of the
thin-layer chromatography in 30% methanol in chloroform showed a yellow
fluorescent spot at R
value of 0.7. Normal butanol
was removed by rotary evaporation, and the solid was purified by
preparative HPLC (
)(isocratic system of 60% 0.01 M ammonium formate and 40% methanol over a Partisil C8 column).
Analytical Data for the
Fluoroarabinoflavin
H NMR data (200 MHz,
Me
SO-d
/trimethylsilane):
8.3 (s, 1H, amide proton), 7.85 (s, 1H, aromatic proton),
7.65 (s, 1H, aromatic proton), 5.5-4.5 (ribityl side
chain protons), 2.4 (s, 3H, aromatic methyl), 2.3 (s,
3H, aromatic methyl).
Positive Ion FAB Mass Spectrometry
Data
(M
+ 1): 379.
F NMR (500
MHz,
Me
SO-d
/C
F
):
66.8 ppm.
Conversion of 2`-F-arabinoflavin to the FAD and FMN
Level
Fluoroarabinoflavin was converted to the FAD level with
partially purified FAD synthetase from Brevibacterium ammoniagenes by incubating the flavin in 0.05 M potassium
P
, pH 7.5, at 37 °C, following the procedure of Spencer et al.(27) . After 24 h, HPLC analysis of the
incubation mixture showed around 40% conversion to FAD and 50% to FMN.
After the addition of a second lot of FAD synthetase, incubation was
continued for a further 24 h. After the second day, 70% of the
riboflavin was converted to the FAD with around 25% contamination by
the FMN. No starting riboflavin was detected. The FAD was purified by
HPLC over an RP-18 column eluted with a mixture of 80% 0.01 M potassium P
, pH 6, and 20% methanol. 2`-F-arabino-FMN
was obtained by hydrolysis of the FAD in 0.1 M potassium
P
, pH 7, with snake venom phosphodiesterase (Naja naja venom).
Synthesis of 2`-Deoxyflavins
The 2`-deoxy
riboflavin was synthesized by the same sequence of reactions described
for the 2`-fluoroarabinoflavin (Fig. S1). In this case
2`-deoxyribose was used in reaction with 3,4-dimethyl aniline as the
first step. Hydrogenation of the Schiff base in the next step was
carried out at room temperature and pressure over Pd/C. The yields of
all the reactions fell in the same ranges as for the 2`-fluoroflavin.
HPLC-pure 2`-deoxyriboflavin was taken to the FAD level with the
partially purified FAD synthetase, again under the same conditions used
for the fluoroflavin. After the second day of incubation, the
riboflavin was converted almost completely to the FAD level.
2`-deoxy-FMN was obtained by treating the HPLC pure 2`-deoxy-FAD with
snake venom.
Positive Ion FAB Mass Spec Data for
2`-Deoxyriboflavin
(M
+ 1): 361.
Synthesis of Arabinoflavins
Arabinoflavin was
synthesized, again by making use of the above method (Fig. S1).
In this case D-arabinose was the sugar used in the first
condensation reaction with 3,4-dimethyl aniline. However the reduction
of the Schiff base obtained in this reaction was found to be very
resistant to hydrogenation and required more drastic conditions. The
reduction was carried out overnight at 60 atm in a Parr hydrogenation
apparatus. The rest of the transformations in the synthesis were
carried out as described for the fluoroflavin. Arabinoflavin was
recrystallized from hot water and used for the reaction with FAD
synthetase. Only about 45% conversion to the FAD level was obtained
even after two incubations with fresh lots of FAD synthetase, and the
rest of the flavin was recovered as the FMN. Arabino FAD was purified
by HPLC (conditions same as fluoroflavin) and then treated with snake
venom to get the pure FMN.
Positive Ion FAB Mass Spec Data for
Arabinoflavin
(M
+ 1): 377.
RESULTS AND DISCUSSION
Fluorescence Properties of the Free Flavins
An interesting descending trend was observed when
quantitative measurements of the fluorescence of the four FAD
molecules, namely normal FAD, 2`-F-arabino-FAD, arabino-FAD, and
2`-deoxy-FAD, were carried out at the same concentration. The order of
fluorescence intensity was determined to be normal FAD >
2`-F-arabino-FAD
arabino-FAD > 2`-deoxy-FAD (1.7:1.4:1.4:1).
It is known that the intramolecular stacking of the adenine moiety on
the isoalloxazine ring of the flavin decreases the fluorescence of FAD
when compared with riboflavin or FMN(28, 29) . When we
made space filling models for these FAD molecules, it was noted that
the substituent at the 2`-carbon restricts the extent of stacking of
the adenine moiety on the isoalloxazine ring. The results suggest a
stronger intramolecular complex between the adenine and isoalloxazine
for the 2`-deoxy-FAD, intermediate for 2`-F-arabino-FAD, or arabino-FAD
and comparatively weaker for normal FAD, in keeping with the size of
the substituent at the 2`-position. This was further substantiated by
converting all of the FAD level molecules to the FMN level with snake
venom and measuring the fluorescence. It was found that the increase in
fluorescence from FAD to FMN for these flavins was 21.5 times for
2`-deoxyflavin, 16.9 times for 2`-F-flavin, 17 times for arabinoflavin,
and 11.3 times for normal flavin. This is a very clear experimental
demonstration of the effect on fluorescence of intramolecular complex
formation between the adenine and isoalloxazine rings in FAD molecules.
Reduction-Oxidation Potentials of Flavins
Redox properties of the flavin and the catalytic properties
of flavoproteins are known to be related. Since we reconstituted the
above flavins into a number of flavoproteins, the measurement of
potentials is of obvious interest. The potentials were measured for
2`-deoxy-FAD and arabino-FAD by using the xanthine/xanthine oxidase
system and anthraquinone-2-sulfonate as the reference dye(30) .
Reduction of the deoxy-FAD has an isosbestic point at 336 nm, and
reduction of the dye has an isosbestic point at 354 nm. These
wavelengths were used to monitor the reduction of the components in a
mixture of the two. A plot of log[ox/red] of the dye against
log[ox/red] of the flavin gave a midpoint potential for
2`-deoxy-FAD of -219.5 mV, slightly more negative than that of
normal FAD (-207 mV; (30) ). Reduction of the arabino-FAD
has also shown an isosbestic point at 336 nm. A similar plot for the
arabino-FAD gave a potential for arabino-FAD of -207 mV,
identical to the value of native FAD. The 2`-fluoroflavin was also
reported to have the same potential as the normal flavin(9) .
F NMR Spectra of Free Flavins
A single peak was recorded for oxidized 2`-F-riboflavin at
66.3 ppm in 0.1 M potassium P
, pH 7. When the
spectrum was recorded for the oxidized form of the free flavin in
dimethyl sulfoxide, a single resonance at 66.8 ppm was recorded. This
suggests very little or no solvent effect on the fluorine chemical
shift, going from protic to aprotic solvent. Sodium dithionite-reduced
2`-F- riboflavin has a signal at 65.5 ppm, around 1 ppm upfield shift
from the oxidized form. It shows, as expected, that reduction of the
isoalloxazine ring has very little effect on the fluorine resonance.
This is unlike the case of 8-F-flavins, where the benzene ring of the
flavin experiences more positive character due to the presence of the
electronegative fluorine (11) and results in large differences
in the chemical shifts of the oxidized (64.9 ppm) and reduced (36.0
ppm) forms. The
F NMR resonance of the oxidized
2`-F-arabino-FAD was seen at 65.2 ppm, one ppm upfield shift from that
of 2`-F-arabinoflavin. This can be attributed to the intramolecular
stacking of the adenine moiety on the
isoalloxazine(28, 29) . The sodium dithionite-reduced
FAD has a peak at 66.1 ppm. The
F resonances for the
oxidized and reduced FMN were recorded at 66.5 and 67.2 ppm
respectively (Table 1).
Riboflavin Binding Protein
The 2`-fluororiboflavin binds to the apoprotein of hen egg
white riboflavin binding protein, with a K
of 4.2
± 0.3
10
M at pH 7, 25
°C. By standardization of the apoprotein with pure riboflavin, the
extinction coefficient at 444 nm of the 2`-fluororiboflavin was
determined as 11,400 M
cm
. The extinction coefficients for 2`-F-arabino-FMN
and 2`-F-arabino-FAD were determined as 11,300 and 10,800 M
cm
, respectively. The
free flavin has 
at 372 and 444 nm. Upon binding to
aporiboflavin binding protein the 
shifted to 378
and 454 nm. The
F NMR spectrum of the oxidized
2`-F-arabino-flavin bound to riboflavin binding protein showed a
resonance at 66.0 ppm. This is the same chemical shift observed for the
unbound free flavin, suggesting minimal protein-fluorine interactions.
However, the increased line width of the signal from that of the free
flavin confirms that the flavin is bound to the protein. When the
reconstituted riboflavin binding protein was reduced with dithionite, a
signal at 65.9 ppm was recorded, again in the same region as the free
flavin. The narrow line width of the signal suggests the flavin may not
be bound to the protein in its reduced state.
Reconstitution of Apolipoamide Dehydrogenase with
Modified Flavins
A small excess (1.5 equivalents) of pure modified flavin was
added to the 50% ethylene glycol solution of apoprotein and incubated
for 24 h at 4 °C. Then, the excess flavin and ethylene glycol were
removed by ultrafiltration with a Centricon 30 microconcentrator and
equilibration with 100 mM potassium P
, pH 7,
containing 0.3 mM EDTA. The reconstituted proteins have well
resolved absorption spectra indicative of an apolar environment, as in
the case of the native enzyme. Comparatively larger shifts from the
free flavin to bound flavin were observed in the spectral region of the
near UV peak (Table 2) in all cases.
Reduction of Reconstituted Lipoamide Dehydrogenase Forms with
Dihydrolipoamide and the Stability of the EH
Form
Native pig heart lipoamide dehydrogenase is reduced
rapidly and almost stoichiometrically by the substrate to the EH
form, and even the use of excess
dihydrolipoamide leads to no further reduction to the EH
form. Dithionite is required for complete reduction of EH
to EH
. Since EH
is the catalytically relevant species, the
effects of the present modifications on the stability of this form were
studied by the anaerobic reduction of the reconstituted proteins with
the substrate (Fig. 2). The typical EH
form
is represented by a charge transfer complex with typical long
wavelength absorption in which the thiolate of Cys-63 is the donor and
FAD is the acceptor(31) .
Figure 2:
Reduction of lipoamide dehydrogenases with
dihydrolipoamide. Reactions were carried out anaerobically in 100
mM potassium P
, pH 7, containing 0.5 mM EDTA at 25 °C. A, reduction of
2`-F-arabino-FAD-lipoamide dehydrogenase with dihydrolipoamide.
-, oxidized enzyme; - - -, after 1.6 eq
of dihydrolipoamide; 


, after 6 eq of
dihydrolipoamide. B, reduction of arabino-FAD-lipoamide
dehydrogenase with dihydrolipoamide. -, oxidized enzyme;
- - -, after 1.6 eq of dihydrolipoamide;



, after 6 eq of dihydrolipoamide. C,
reduction of 2`-deoxy-FAD-lipoamide dehydrogenase with
dihydrolipoamide. -, oxidized enzyme; - -
-, after 1.6 eq of dihydrolipoamide; 


,
after 6 eq of dihydrolipoamide.
Arabino-FAD-Lipoamide Dehydrogenase
The spectrum
recorded immediately after the anaerobic addition of 1.6 eq of
dihydrolipoamide to the reconstituted protein showed little or no
charge transfer band typical of the EH
form, with
about 50% reduction in the flavin absorption bands. After this spectrum
was stable, addition of another 4 eq of substrate resulted in complete
reduction; the spectrum recorded is typical of the EH
form (Fig. 2A).
2`-F-Arabino-FAD-Lipoamide Dehydrogenase
The
absorption spectrum of the reconstituted protein with 1.6 eq of
substrate, showed somewhat less long wavelength absorbance than is
typical for the EH
form. With the addition of
another 6 eq of substrate, further reduction of the flavin absorption
was observed, but with the development of more long wavelength
absorbance (Fig. 2B).
2`-Deoxy-FAD-Lipoamide Dehydrogenase
With 1.6 eq
of substrate, the enzyme was reduced to the EH
form with the typical charge transfer band. Further addition of
another 6 eq of dihydrolipoamide resulted in the slow reduction of the
flavin absorption peaks with a decrease in the charge transfer band,
suggesting a small reduction to the EH
form (Fig. 2C).These results suggest that the
modifications at the 2`-position have affected the stability of the EH
form of the protein, presumably by changing the
relative redox potentials of the bound flavin and the active site
disulfide/dithiol couple. The effect is clearly most dramatic with
arabino-FAD enzyme.
Reduction of 2`-F-arabinolipoamide Dehydrogenase with
NADH
For the native enzyme, the spectrum when reduced by
stoichiometric NADH is slightly different from that when reduced by
dihydrolipoamide. This is because of the formation of an EH
NAD
complex when NADH is
the reductant(32) . 2`-F-arabino-FAD-reconstituted protein was
reduced anaerobically with NADH (Fig. 3A). The spectrum
resulting from the addition of one equivalent of NADH shows much less
absorbance in the 530 nm region than the typical EH
form of the enzyme. The extent of bleaching at 450 nm is
indicative of the presence of an appreciable amount of flavin in the
fully reduced form. After no further changes were observed spectrally,
another 2 eq of NADH was added. This resulted in the complete reduction
of the flavin, and the charge transfer band of reduced flavin with
NAD
(33) was observed.
Figure 3:
Anaerobic reduction of
2`-F-arabinolipoamide dehydrogenase with NADH and NADPH. Reactions were
carried out in 100 mM potassium P
, pH 7,
containing 0.5 mM EDTA at 25 °C. A, reduction
with NADH. -, oxidized enzyme; - - -,
after addition of 1 eq of NADH; 


, after
addition of another 2 eq of NADH. B, reduction with NADPH.
-, oxidized enzyme; - - -, adding 2 eq
of NADPH; 


, after further addition of 1 eq of
NAD
.
Reduction of 2`-F-arabinolipoamide Dehydrogenase with
NADPH
Lipoamide dehydrogenase is very selective for NADH, and
NADPH is not a catalytically competent substrate. However, it results
in the slow reduction of the enzyme without complications arising from
binding of the oxidized pyridine nucleotide that occur with NADH as
reductant(33) . Since we found that the reduction of
2`-F-arabinolipoamide dehydrogenase with NADH is quite facile and
results in further reduction from EH
with very
little excess, reduction with NADPH was studied. 2`-F-arabino-FAD
enzyme was reacted anaerobically with 2 eq of NADPH, and the reduction
was followed spectrophotometrically (Fig. 3B). The
final spectrum showed a charge transfer band typical of EH
, which was stable over a period of several
hours. When one eq of NAD
was added at this point, it
resulted in the facile reduction of the flavin with a spectrum typical
of the charge transfer complex between reduced flavin and
NAD
(33) . These results suggest that
NAD
binding influences the interaction between the
flavin and the reduced disulfide in this modified protein, presumably
by altering the relative oxidation-reduction potentials of the flavin
and active center disulfide such that reduced flavin is more
thermodynamically stabilized than in the native enzyme, even in the EH
form. In the case of native enzyme,
NAD
appears to function as an effector by reacting
with 4-electron reduced enzyme (EH
) to produce EH
. This effectively increases the concentration
of EH
in the steady
state(34, 35, 36, 37) .
F NMR Studies
Oxidized and Dihydrolipoamide Reduced
Proteins
The oxidized form of the protein (E) has one
F resonance at 72.3 ppm, an
8-ppm downfield shift
from that of the free flavin (Fig. 4). To the same sample, 4 eq
of dihydrolipoamide was added in situ after flushing the tube
with argon for 15 min. The
F NMR spectrum of this reduced
protein (EH
) has a very broad resonance between
71.5 and 72.5 ppm, which is essentially the same chemical shift as the
oxidized form (Fig. 4).
Figure 4:
F NMR spectra for
2`-F-arabino-FAD-lipoamide dehydrogenase and its reduced forms. Spectra
were recorded in 100 mM potassium P
, pH 7,
containing 0.5 mM EDTA at 22 °C and hexafluorobenzene as
an external standard. Reductants were added after flushing the NMR
tubes above the sample solution with argon for 15 min. The enzyme
concentration was 110 µM. A, oxidized form of
2`-F-arabino-FAD-lipoamide dehydrogenase; B, after reducing
with 3 eq of dihydrolipoamide; C, after reducing with 3 eq of
NADPH; D, after reducing with excess sodium
dithionite.
NADH-reduced Protein Forms
F NMR
spectra were recorded for various forms of the 2`-F-FAD-reconstituted
lipoamide dehydrogenase (Fig. 5) by reducing with NADH. The
chemical shifts for the three different forms of the protein showed
marked differences. The protein was mixed with 3 eq of NADH in the NMR
tube after flushing with argon. The NMR spectrum of this sample showed
only a very weak signal at 79.1 ppm, a further downfield shift of 7
ppm. We were able to obtain a reasonably good signal by changing the
recycling time to 0.005 s. Although the spectral results reported in Fig. 4indicate full reduction of the enzyme flavin with 3 eq of
NADH under strict anaerobic conditions, the color of the enzyme in the
NMR tube was pink, indicating the presence of the EH
NAD
complex, probably due
to difficulty of achieving strict anaerobiosis in the NMR experiments.
Presumably we recorded the spectrum for the EH
NAD complex in this case. When the protein
was reduced to the EH
form by adding excess NADH,
a signal was recorded at 64.8 ppm, an upfield shift of 12 ppm from the EH
form to almost the free flavin region.
Reduction with excess NADH results in the formation of reduced flavin
in complex with NAD
and is characterized by the
blue-green color of the complex(33) . The results suggest that,
unlike in the E or the EH
forms, the
fluorine might not be interacting with the protein in the EH
form.
Figure 5:
F NMR spectra for NADH
reduced 2`-F-arabino-FAD-lipoamide dehydrogenase. Spectra were recorded
in 100 mM potassium P
, pH 7, containing 0.5 mM EDTA at 22 °C and hexafluorobenzene as an external standard.
NMR tubes above the sample solution were flushed with argon for 15 min
prior to the addition of the reductant. The enzyme concentration was
120 µM. A, after reducing with 2.5 eq of NADH; B, after reducing with 12 eq of
NADH.
NADPH-reduced Protein
When the protein was reduced
with dihydrolipoamide to obtain the spectrum for the uncomplexed EH
form, the NMR spectrum showed one broad signal
between 71.5 and 72.5 ppm. Because of the problems of establishing
strict anaerobiosis in the NMR experiments, there was a possibility
that we might be obtaining the NMR spectrum of the oxidized form of the
protein. Hence
F NMR was recorded with the NADPH-reduced
protein since it is known that EH
does not form a
complex with NADP
, and hence one should obtain the NMR
spectrum for the ligand-free EH
form. This
spectrum showed two clear well resolved resonances at 71.3 and 72.3 ppm (Fig. 4). One of these is the same as the oxidized form of the
protein and the other at 71.3 is presumably from the EH
form. This is in agreement with the spectrum obtained from the
dihydrolipoamide-reduced protein and suggests relatively small changes
in the fluorine chemical environment from oxidized to the EH
form.
Dithionite-reduced Protein
The NMR spectrum that
we obtained by reducing the fluoroarabino-FAD enzyme with excess NADH
is that of the complex between reduced flavin and NAD
.
To see if the complex formation of EH
with NAD
perturbed the
F chemical shift, NMR was recorded for the
ligand-free EH
form by reducing the protein with
excess dithionite. Dithionite-reduced enzyme showed a single resonance
at 67.8 ppm, around 3 ppm upfield shift from that of the complex (Fig. 4).
Steady State Kinetics
All three reconstituted
forms of the protein showed parallel line double-reciprocal plots when
the NAD concentration was varied over several concentrations of
dihydrolipoamide, as in the case of the native enzyme(37) . The
results (Table 3) show that the catalytic activity of the protein
is seriously hampered by the present modifications at the 2`-position
of the ribityl side chain. The 2`-deoxy-FAD form of the protein was
found to be relatively more active than both the arabino forms. Rapid
reaction kinetics studies are planned to determine which steps in
catalysis are altered in the modified enzymes.
Reconstitution of Apoglutathione Reductase
The apoprotein was reconstituted with the three modified
flavins by incubating with 1.5 eq of the flavin for 90 min at 0 °C
and then the excess flavin was washed off with 100 mM
potassium P
pH 7 containing 0.3 mM EDTA over a
Centricon 30 microconcentrator. Absorption spectra of the reconstituted
proteins showed the shifts typical of the native protein from the free
flavin. In distinction to lipoamide dehydrogenase, little or no shift
in the near UV flavin peak was observed on binding to the protein (Table 2).
Reduction of the Reconstituted Glutathione Reductase Forms with
DTT and the Stability of the EH
Form
The spectrum of
two-electron reduced glutathione reductase is very similar to that of
lipoamide dehydrogenase and represented by the typical charge transfer
band. DTT reduces the native protein to its EH
form. The reconstituted proteins were reduced anaerobically with
DTT, and reduction was followed spectrophotometrically (Fig. 6).
Arabino-FAD-reconstituted protein showed a typical EH
spectrum immediately after mixing with DTT but was gradually
reduced to the EH
form with concomitant decrease
in the charge transfer band (Fig. 6A). The
2`-F-arabino-FAD reconstituted protein after mixing with DTT (final
concentration, 100 µM) formed a typical charge transfer
band. Since no further spectral changes were observed after 1 h, more
DTT was mixed (final concentration, 1 mM). This resulted in
the complete reduction of the flavin, presumably to EH
form, in 3 h (Fig. 6B). In contrast, the
reduction of 2`-deoxy-FAD-reconstituted protein resulted in a quite
stable EH
form with no further reduction to EH
(Fig. 6C).
Figure 6:
Anaerobic reduction of glutathione
reductases with DTT. Reactions were carried out in 100 mM potassium P
, pH 7, containing 0.5 mM EDTA at
25 °C. A, reduction of arabino-FAD-glutathione reductase
with DTT (final concentration, 100 µM). -,
oxidized enzyme; - - -, 15 s after the addition of
DTT; -
-
-, after 45 s;
-

-, after 90 s; 


,
after 180 s. B, reduction of 2`-F-arabino-FAD-glutathione
reductase with DTT; -, oxidized enzyme; - -
-, stable spectrum after the addition of DTT (final concentration
100 µM); -
-
-, after the
addition of excess DTT (final concentration 1 mM). C,
reduction of 2`-deoxy-FAD-glutathione reductase with DTT.
-, oxidized enzyme; - - -, stable
spectrum after the addition of DTT (final concentration, 100
µM).
F NMR Studies
The
2`-F-arabino-FAD-reconstituted glutathione reductase has a resonance at
73 ppm, which is essentially in the same position as for the
corresponding lipoamide dehydrogenase and mercuric reductase (Table 1). This suggests that in these proteins the fluorine
substituent is experiencing the same chemical environment, since the
resonances with unrelated flavoproteins vary in the range 65-81
ppm. (
)While the protein can be reduced to the EH
form by adding DTT, unfortunately no NMR signal
could be observed, even with prolonged data collection times.
Effect of DTT on the
F NMR Spectrum of Free
Flavin
The 2`-F-arabinoflavin was found to be stable to
nucleophilic displacement reactions with sulfide, normal thiols, and
reduction with dithionite. In the light of the above effect of DTT on
the NMR spectrum of reconstituted glutathione reductase, the effect on
the free flavin was studied in detail. The
F NMR spectrum
was immediately quenched on addition of DTT to the fluoroflavin in 50
mM potassium P
, pH 7. After several thousand
scans, a less intense signal was recorded (5000 scans with exponential
line broadening 100 Hz) when compared with pretreated flavin (50 scans
with exponential line broadening of 10 Hz), with slow fluoride
elimination. After 24 h, a peak at 46.9 ppm, which corresponds to free
fluoride, was recorded. Efforts to purify the flavin product and
characterization by FAB mass spectrometry failed, as the product was
found to be extremely labile. No spectral perturbations were observed
spectrophotometrically on adding DTT to the flavin. It was found that
the addition of mercaptoethanol has no effect on the NMR spectrum, and
no fluoride elimination was observed over the same time scale. To get
more insight into this observation, the effect of DTT on the NMR in an
aprotic solvent was also studied. When DTT was added to the flavin in
Me
SO, neither was there any effect on the NMR spectrum nor
was there any fluoride release. This clearly suggests that DTT in its
ionic form might be complexing with the flavin side chain, thereby
facilitating the release of fluoride by another DTT molecule. This was
further supported by repeating the aqueous experiment at pH 10.5, where
the effect was more pronounced and fluoride elimination was relatively
faster. The signal for the released fluoride was observed within a few
minutes, unlike at pH 7, where it took a few hours. With
mercaptoethanol as the nucleophile at this pH, very slow release of
fluoride was observed over a period of a few days.In the light of
the above observations, it was of obvious interest to check if DTT has
a similar effect on the
F NMR spectra of other fluoro
compounds. Hence
F NMR spectra of 4-F-phenol and
2,4-difluorophenol were recorded in the absence and presence of DTT.
The 4-F-phenol shows one resonance at 41.6 ppm. When DTT was added, no
effect was found on the NMR signal and no fluoride release was observed
over a period of a few hours. The 2,4-difluorophenol has two peaks at
44.4 and 33.3 ppm, and again DTT addition resulted in no effect on the
spectrum and in no fluoride elimination.
Further Reduction of the DTT-reduced
2`-F-arabino-FAD-glutathione Reductase with Dithionite; Elimination of
Fluoride
Since it was not possible to observe the
F
NMR spectrum for the DTT-reduced protein, it was further reduced to EH
with dithionite, and the NMR spectrum was
recorded again. Interestingly, slow elimination of fluoride was
observed with no signal visible for the flavin. When the protein was
denatured with SDS, no flavin covalently bound to the protein was
observed, as all of the flavin was found in the ultrafiltrate on
centrifuging through a Centricon 30 microconcentrator. In a separate
experiment the DTT-treated protein was reoxidized by removal of the
excess DTT and washing with buffer in a Centricon 30 microconcentrator.
This reoxidized sample showed the same
F resonance as the
initial 2`-F-arabino-FAD enzyme, consistent with no fluoride
elimination being detected in the NMR of the DTT-treated protein,
whereas dithionite addition in presence of DTT resulted in the release
of fluoride. It should be noted that free 2`-F-arabino-FAD and several
2`-F-arabino-FAD-reconstituted proteins on reduction with dithionite
showed no fluoride elimination over a period of weeks, (
)whereas treatment of the free flavin with DTT resulted in
slow fluoride elimination.
Activities of the Reconstituted Glutathione Reductase
Forms
Standard aerobic assays were carried out with the
reconstituted proteins in 100 mM sodium P
, pH 7.6,
containing 0.3 mM EDTA, at 25 °C. The turnover numbers
suggest that the activities are hampered in case of arabino- and
fluoroflavin- reconstituted forms, but with relatively small effect on
the 2`-deoxy-FAD-reconstituted form (Table 4). Since the
stabilities of the EH
form of the reconstituted
proteins were found to vary, it was thought that the impairment of the
activities might be due to the accumulation of the EH
form during catalysis. This possibility was tested by the
NADPH-thionicotinamide NADP transhydrogenase activity of the native as
well as the reconstituted proteins (Table 4). The results are in
accord with the spectral observations that the EH
form of the protein was destabilized in case of
arabino-FAD-reconstituted glutathione reductase and suggest that the
protein may exist significantly in the EH
form
during catalytic turnover. This could result in reduced catalytic
activity in the NADPH-GSSG reductase assay, but increased activity in
the transhydrogenase assay, which relies only on reduction and
oxidation of the flavin. Again rapid reaction studies will be necessary
to determine which steps in catalytic turnover are affected by the
flavin modifications.
Reconstitution with Apomercuric Reductase
Mercuric
reductase is a dimer containing one FAD/monomer and shares many common
spectral features with lipoamide dehydrogenase and glutathione
reductase. A charge transfer interaction between an active center
thiolate and oxidized FAD gives two-electron reduced EH
a characteristic absorption spectrum as in case of the other two
proteins(38) . Studies with the 2`-F-arabino-FAD reconstituted
mercuric reductase (9, 39) showed that DTT reduces the
flavin with no stabilization of the EH
form,
whereas the native protein is stabilized as the two-electron reduced EH
form when treated with DTT. Hence it was of
obvious interest to determine the effect of arabino-FAD as well as
2`-deoxy-FAD on the stability of this catalytically important form of
the protein. Apoprotein was reconstituted by incubating with 1.2 eq of
modified flavins at 0 °C for 60 min. Excess flavin was washed off
with 50 mM potassium P
, pH 7, over a Centricon 30
microconcentrator. The binding of these flavins was accompanied by
around 6-8-nm blue shift of the flavin absorption maximum (Table 2). Comparatively larger shifts from the free flavin were
observed in the spectral region of the near UV peak in the case of the
fluoroarabino-FAD enzyme.
Reduction of Reconstituted Mercuric Reductase Proteins
with DTT; Stability of EH
Form
The arabino-FAD
reconstituted mercuric reductase was reduced with excess DTT, and the
reduction was followed spectrophotometrically (Fig. 7A).
Reduction of the flavin is observed with no detection of the charge
transfer band typical of the EH
form, similar to the
results reported for the 2`-F-arabino-FAD enzyme(9, 39) When the 2`-deoxy-FAD-reconstituted protein was reduced
with DTT, slow but full development of the charge transfer band for the EH
form was obtained and found to be stable over a
period of several hours without any further reduction (Fig. 7B).
Figure 7:
Reduction of mercuric reductases with DTT.
Reactions were carried out in 50 mM potassium P
,
pH 7, at 25 °C. A, reduction of arabino-FAD-mercuric
reductase with DTT; -, oxidized form of the enzyme;
- - -, 15 s after addition of 5 mM DTT;



, 30 min after the addition of DTT. B,
reduction of 2`-deoxy-FAD-mercuric reductase with DTT; -,
oxidized form of the enzyme; - - -, 10 min after
addition of 5 mM DTT; -

-, 120
min after the addition of DTT.
F NMR Studies
F NMR
spectra for the oxidized form of the 2`-F-arabino-FAD reconstituted
mercuric reductase showed a signal at 72.3 ppm, which is in the same
position as for the other two members of the family, suggesting closely
similar chemical environments experienced by the fluorine in all these
proteins (Table 1).
Activities of the Reconstituted Mercuric Reductase
Forms
Standard aerobic assays were carried out with all the
reconstituted proteins in 50 mM potassium P
, pH
7.3, at 37 °C (Table 5). The turnover numbers suggest that
the activities are significantly decreased for all the reconstituted
proteins. Reconstitution of lipoamide dehydrogenase and glutathione
reductase with 2`-deoxy-FAD resulted in relatively active forms when
compared with the arabino-FAD reconstituted forms. In the case of
mercuric reductase, although the EH
form is
stabilized, the 2`-deoxy-FAD enzyme is as inactive as the other two
forms.
CONCLUSIONS
The present studies have shown that modification at the 2`
position of the ribityl side chain has a pronounced effect on the
catalytic activities of this family of disulfide oxidoreductases.
Reconstitution of the proteins with arabino FAD destabilizes the EH
form and the accumulation of the EH
form during catalysis is suggested. This
concept is supported by the transhydrogenase activities of the
glutathione reductases. The destabilization of the EH
form could be attributed to the absence of the 2`-hydroxyl group,
which stabilizes the thiolate of the EH
form in
the native enzyme(3) . However, it is interesting to note that
the deoxy-FAD- and the fluoro-FAD-reconstituted proteins result in
relatively stable EH
forms. It is possible that
the EH
form might be stabilized by a solvent
molecule through a hydrogen bond, which can take the place of the
hydroxyl group. During catalysis, the formation of the EH
form is excluded in the native FAD proteins by
the fact that the EH
/EH
reduction potentials are lower than that for
NADP/NADPH(35, 10) . Although the redox potentials of
the free modified flavins are essentially the same as native FAD, the
effect of binding at the active site might alter them in such a way
that they can influence catalysis. Results obtained from the pyridine
nucleotide reduction experiments suggest that NAD
binding influences the interaction between the flavin and the
reduced disulfide in the 2`-F-arabino-FAD-lipoamide dehydrogenase,
presumably by altering the relative oxidation-reduction potentials. It
is unlikely, however, that the effects on catalytic activity can be
rationalized solely on the thermodynamic stability of the EH
forms, since with lipoamide dehydrogenase, all
three modified enzymes have much lower activities than native enzyme,
despite some of them having thermodynamically stable EH
forms. A similar lack of correlation is found
with mercuric reductase. Detailed rapid reaction kinetics studies will
be required to probe these questions further. The studies have provided
F NMR data for different forms of the lipoamide
dehydrogenase, which suggest marked conformational changes from one
form to the other. The data for the oxidized form of all the three
proteins of this family suggest that the fluorine experiences
chemically very similar environments at the active site. As
anticipated, the 2`-F-arabinoflavin showed marked stability toward
general nucleophiles like sulfide, hydroxide, normal thiols and
reduction with sodium dithionite. However, the elimination of the
fluorine substituent observed in presence of DTT suggests that the
fluorine is susceptible to nucleophilic attack by very reactive
reagents.