2411
J. Phys. Chem. 1991, 95, 241 1-2415 correction is only partially cancelled by the higher order corrections determined in the coupled cluster procedure. In spite of such a cancellation, the cumulative electron correlation contribution is large and amounts to almost 40% of the tautomer relative stability. This seems to suggest that one cannot obtain a correct relative energy of tautomers of 2( 1H)-pyridinethione at the lower orders of the many-body perturbation theory because of cancellations of lower order terms with higher order terms. We conclude that the large contribution to the relative stability of the tautomers arising from the low-order (e.g., from second order) perturbation theory may be considerably reduced by higher order terms. As a consequence, the relative stability estimated with the S C F + scaled ZPE MBPT(2) higher order coorelation methods would fall closer to the experimentally derived values than the MBPT(2) result, as is probably true for diazinethiones. However, the inclusion of the higher order correlation terms does not seem to be enough to compensate for the systematic discrepancy between the theoretical and the experimental estimates of the relative stability of the tautomers.
difference was similar (AF 4.5 kJ/mol). 3(2H)-Pyridazinethione adopts exclusively the thione form in inert gas matrices. Upon UV irradiation, the photoinduced isomerization to thione occurs in the matrix isolated samples of 4(3H)-pyrimidinethione and 3(2H)-pyridazinethione. The IR spectra of the thione and thiol tautomers were predicted theoretically at the SCF/3-21G* level, and a comparison of the predicted and experimentally observed spectra gave an assignment of most of the observed bands. The theoretical a b initio evaluation of the thiol/thione concentration ratio falls closer to the experimental estimate when the higher order electron correlation effects are taken into account. However, the discrepancy between the theoretical and experimental estimates of the relative stability of the tautomer's still persists.
Conclusions
Supplementary Material Available: The internal coordinates (Table IV and VII), experimental frequencies, integral intensities, and assignment to the normal modes (Tables V, VI, VIII, IX) for 4S-pmd, 4HS-pmd, 3S-pdz, and 3HS-pdz tautomers; details of the ab initio calculations for 2(1H)-pyridinethione (Table X); IR spectra of N z matrix isolated 4(3H)-pyrimidinethione and 3(2H)-pyridazinethione (Figures 6 and 7) (1 5 pages). Ordering information is given on any current masthead page.
+
+
The gas-phase and matrix isolation experiments showed that 4(3H)-pyrimidinethione exists, under conditions where intermolecular interactions are minimized, as a mixture of thiol and thione tautomers. The relative concentration of tautomers, in the gas phase ([thiol]/[thione] 3 at 483 K) and in matrix experiments ([thiol]/[thione] 5 at 350 K), depends on the temperature. In both cases, the experimentally derived value of the free energy
- -
N
Acknowledgment. This study was supported, in its experimental part, by the grants C.P.B.P.Ol.12. and C.P.B.R.ll.05. provided by the Polish Academy of Sciences.
Kinetics of the Oxidation of NADH by Methylene Blue In a Closed System Peter Sevcikt and H. Brian Dunford* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6C 2C2 (Received: July 16, 1990; In Final Form: October 19, 1990)
The kinetics of anaerobic oxidation of nicotinamide adenine dinucleotide (NADH) by methylene blue was investigated in phosphate and glycine buffers with an excess of NADH in a closed system. The reaction is first order with respect to the concentration of methylene blue. The observed rate constant is pH independent over the pH range of 7.0-10.6 and increases with NADH concentration in a saturated mode. The oxidation process was also investigated under aerobic conditions in a closed system. Applying the steady-state approximation to methylene blue, the rate constant for reoxidation of the reduced form of methylene blue by oxygen was estimated to be 1.62 X IO2 M-' s-I at pH 9.0 at 25 OC. The implications of the present results for NAD' regeneration and oscillatory reactions are noted.
Introduction The aerobic oxidation of nicotinamide adenine dinucleotide (NADH) catalyzed by the enzyme horseradish peroxidase (HRP) is known to exhibit damped oscillations in a closed system.' When this reaction is carried out in an open system in which oxygen and/or NADH are continuously supplied to a well-stirred solution containing HRP, methylene blue (MB'), and 2,4dichlorophenol (DCP), a variety of nonlinear behavior can be observed including bistability2 (even in the absence of MB' and DCP), chaos,3 and sustained oscillations.4 Sustained oscillations started immediately after MB+ was added to a steady-state reaction mixture of NADPH, O2 and lactoperoxidase.5 Analysis and computer simulations of some proposed models and mechanisms causing oscillations with NADH have been performed.*" However, the mechanistic role played by MB+ in the sustained peroxidase-catalyzed oscillations is not clear and needs to be e l ~ c i d a t e d . ~According *~J~ to Olsen and Degn,4 the role of MB+ 'Permanent address: Department of Physical Chemistry, Comenius University, 842 15 Bratislava, Czechoslovakia.
must be catalytic since the total amounts of O2 and NADH consumed greatly exceed the amounts of MB' in the solution. Meantime, Burger and FieldIz discovered oscillations during the methylene blue catalyzed oxidation of sodium sulfide by 02. The catalyst and O2oscillate only in a continuous-flow stirred tank reactor (CSTR). Oxygen is present in relatively low concentration; it is essentially consumed in one phase of an oscillation and must ( I ) Yamazaki, I.; Yokota, K.; Nakajima, R. Biochem. Biophys. Res. Commun. 1965, 21. 582. (2) Degn, H. Nature 1968, 217, 1047. (3) Olsen, L. F.; Degn, H. Nature 1977, 267, 177. (4) Olsen, L. F.; Degn,H. Biochim. Biophys. Acra 1978, 523, 321. (5) Nakamura, S.; Yokota, K.; Yamazaki, 1. Nature 1%9, 222, 794. (6) Yokota, K.; Yamazaki, I. Biochemistry 1977, 16, 1913. (7) Olsen, L. F. Biochim. Biophys. Acta 1978, 527, 212. (8) Fedkina, V.; Ataullakhanov, F.; Bronnikova,T. Biophys. Chem. 1984, 19, 259. (9) Aguda, B. D.; Clarke, B. L. J . Chem. Phys. 1987,87, 3461. (10) Aguda, B. D.; Larter, R. J . Am. Chem. SOC.1990, 112, 2167. ( 1 1) Alexandre, S.; Dunford, H. B. Manuscript in preparation. (12) Burger, M.; Field, R. J. Nature 1984, 307, 720.
0022-3654/91/2095-241 1%02.50/0 0 1991 American Chemical Society
2412 The Journal of Physical Chemistry, Vol. 95, No. 6. 199
Sevdk and Dunford
SCHEME I
1.01
I
I
I
800
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e
I a
0.4
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0.2
-
be replenished by the flow for the cycle to continue. The oxidation of HS- by MB+ in the absence and presence of oxygen are the most complex parts of the mechanism for ~scillations.'~J~ The reaction orders for [MB+] of 0.5 and for [HS-] 1.5 were found in the absence of 0,and rationalized by a radical-chain mechanism.I4 The kinetic study of the oxidation of NADH by MB+, which we present in this paper, seems of interest in view of (a) the application of MB+ as a potential catalyst in aerobic oscillating reactions; (b) the analytical use in the determination of NADH activity; NADH is very important in biochemical analysis since many enzyme and substrate assays are coupled to the formation or consumption of NADH which is usually followed by spectrophotometry, fluorescence, or chemiluminescence methods; (c) the function of MB+ as a bypass in the respiratory chain in the case of cytochrome poisoning, where MB+ serves as a mediator in the transfer of a pair of electrons from NADH to molecular oxygen. The use of MB+ for the regeneration of NAD+ from NADH, which may be important not only in analytical assays but in preparative enzymatic syntheses, is based upon the reactions shown in Scheme I.15J6
Experimental Section Generol. NADH was purchased from Sigma as a disodium salt. Methylene blue (MB+) was purchased from Fisher. Water was distilled and purified by a Milli-Q water purification system (Millipore). Anaerobic conditions were obtained by bubbling nitrogen gas, purified by passage through an Oxi Clear disposable gas purifier, Model No. DGP-250-R1, into buffers and reactant solutions. All other chemicals were either analytical or reagent grade and used without further purification. The following buffers were used: phosphate prepared from NaH2P04and Na2HP04 (0.1 M, pH 7.02-8.03) or glycine prepared from glycine and NaOH (0.1 M, pH 8.59-10.56) by weight. The ionic strength of glycine buffer was maintained a t 0.1 M with Na2S04. The pH values were determined with a Fisher digital pH meter Model 420 equipped with a Fisher Microprobe combination glass electrode. The pH meter was referenced each time before use with buffered standard solutions a t pH = 7.00, 8.00, and 9.00 purchased from Fisher. Ultraviolet-visible spectra were taken on a Cary 2 19 spectrophotometer, equipped with thermally jacketed cuvette holder, using l c m quartz cells at 25.0 f 0.2 OC. Stock solutions of NADH and MB+ were made according to their nominal molecular weight and the final concentrations determined by measuring the absorbances of diluted solutions at 340 nm (t = 6.22 X IO3 M-' cm-I) and 665 nm (e = 7.8 X lo4 M-' cm-I), respectively. Our own determination of these t values is in agreement with those in the literature.16 Stock solutions of NADH (-2 X lo-, M) in buffer (0.1 M, pH 9.0) were stored under N, at 25 'C in the dark and never used for more than 3 h. All stocks of MB+ M) in buffer (0.1 M, pH = 9.0) (13) Resch, P.; Field, R. J.; Schneider, F. W. J . Phys. Chem. 1989, 93, 2183. (14) Resch, P.;Field, R. J.; Schneider, F. W.; Burger, M . J . Phys. Chem. 1989, 93, 8181. (15) Williams 111, D.C.;Seitz, W.R. Anal. Chem. 1976,18, 1478. (16) Lee, L. G.; Whitwides, G. M.J. Am. Chem. Sa.1985,107,6999.
O'
&
I
370
'
880
Wavelength (nm)
Repetitive scan of absorbance for the reaction of NADH with MB+ in 0.1 M glycine buffer at pH 9.0 and 25 OC under anaerobic M: (a) conditions. [NADHIo = 1.51 X 104M; [MB+l0= 1.08 X taken immediately after mixing; (b-e) taken at subsequent 20-min intervals. Figure 1.
were kept under N2 at 25 OC in the dark and used within one day. Kinetics. The choice of experimental conditions was influenced by the spontaneous decomposition of NADH.6 We have determined the rate constant for this spontaneous reaction to be k,, = 1.13 X s-l in phosphate buffer (0.1 M, pH = 7.02), k, = 2.71 X 10" s-I in phosphate buffer (0.1 M, pH = 8.03), a n i k,, < 1.0 X 10" s-l in glycine-NaOH buffer (0.1 M, pH = 9.0) a t 25 O C . We never observed any measurable decomposition of MB+ in buffers in the absence of NADH. In all cases, conditions were chosen so that such background reactions occurred to an extent of less than 1% of the rate of the reduction of MB+ by NADH. A typical kinetic procedure is as follows. To a cuvette (3.5 mL) containing buffer (1.9 mL) was added NADH (0.08 mL, 1 mM in the cuvette). The cuvette was capped with a rubber septum. Inlet and outlet needles were attached, and nitrogen was bubbled through the solution for at least 15 min (200 mL of N2/min). The cuvette was placed in a cell holder and thermostated. After the temperature had reached 25 O C (usually in 3-4 min), previously deoxygenated MB+ was injected into the cuvette (0.02 mL, 0.01 mM in the cuvette) which was inverted several times to mix the contents. All reactions were conducted under pudo-first-order conditions with 14- to 300-fold excess of NADH over MB+. Reactions were followed by the decrease in the absorbance due to MB+ at 665 nm. At this wavelength none of the other reactants or products has any measurable absorption. Values of kh were obtained from at least two measurements and calculated from plots of In & / A ) versus time. Each run showed good pseudo-first-order kinetics for at least 90% of the reaction, indicating there are no complications resulting from accumulation of reaction products or traces of oxygen in reaction solutions. The absorption spectrum of the solution after completion of the reaction shows exact agreement with a separately prepared solution of NADH (in the absence of oxygen) or a solution of MB+ (in the presence of an excess of 0,) of the same concentrations. The standard error of the rate constants was 6% in glycine buffer and 8% in phosphate buffer. Results The principal difference in the behavior of an anaerobic and an aerobic solution of NADH with MB+ is illustrated in Figures 1 and 2. When a solution of 1.51 X lo4 M NADH is allowed
The Journal of Physical Chemisrry, Vol. 95, No. 6,1991 2413
Oxidation of NADH by Methylene Blue
o.8
1 .o
:I
0.7
0.8
8
0.6
0.8
t8i
'
0
0.4
0.2
I c 330
I
370
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I
I
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880
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Wavelength, nm
Figure 2. Overlay spectra of a solution of NADH (1.5 1 X IO4 M) and MB+ (9.95 X IOd M) at pH 9.0 (0.1 M glycine buffer) and at 25 O C under aerobic conditions (a) taken immediately after mixing; (b-f') taken at subsequent 30411 intervals. to react with 1.08 X M MB+ in 0.1 M glycine buffer at pH 9.0 under anaerobic conditions, the absorption spectrum of the reaction solution shows no significant change in the absorption region of the NADH moiety at 320-380 nm, while the absorption maximum of MB+ at 665 nm gradually decreases during the reaction. Due to finite and increasing absorption of the MB+ in the region below 350 nm, a slight increase in absorption below 350 nm and a shift of the absorption maxima to 336 nm can be observed immediately after mixing of the reactants (Figure 1, curve a) in comparison to the spectrum of the NADH (similar to curve e in Figure I ) . The resultant spectrum contains contributions from both NADH and MB+. Under aerobic conditions (the initial oxygen concentration corresponded to the air-saturated solution), the absorption spectrum of the reaction solution (initially 1.51 X lo4 M NADH and 9.95 X lo4 MB+ in 0.1 M glycine buffer at pH 9.0) shows a decrease of the NADH concentration, while there is no observable change in the absorption region of MB+ at 580-700 nm. These results demonstrate that NADH is smoothly oxidized by MB+ and, if molecular oxygen is present in the reaction solution, the leucomethylene blue produced is much more rapidly reoxidized to MB+. In this electron transport chain, MB+ operates as a reversible electron acceptor-donor which results in the transfer of one hydride equivalent from NADH to molecular oxygen. Even in the presence of a 15.3-fold excess of NADH, the concentration of MBt remained unchanged during the reaction. Due to this invariance of the MB+ concentration in the presence of 02,oxidation rates of NADH can be determined under pseudo-first-order conditions by following the decrease of the absorption at 336 or 340 nm. A plot of In (A,, - A,) X (A, - AJ-I versus time gives a good straight line which passes through the origin. From the data in Figure 2 the observed first-order rate constant kobE= 5.18 X IO-$ s-I and the second-order rate constant k2 = kobs/[MB+] = 5.21 M-I s-' have been calculated for oxidation of NADH by MB+ a t pH 9.0 and 25 OC. At substantially higher concentrations of NADH and/or at lower partial oxygen pressure, deviations are observed and pseudo-first-order conditions are therefore not fulfilled. Figure 3 shows the absorbance a t 665 nm due to MB+ after (2.50-10.2) X lW3 M air-saturated solutions of NADH are mixed with an air-saturated solution of MB+ at pH 9.0 (0.1 M glycine buffer). For the conditions shown in Figure 3A, the capped cuvette with 2 mL
I
I
I
20
40
80
0
Time, min
Figure 3. Time dependence of the absorbance of MB+ at 665 nm in the reaction with NADH at pH 9.0 (0.1 M glycine buffer) and 25 OC in the presence of oxygen. Initial concentrations: [MB+l0 = 1.01 X IOd M; [O2lO= 2.4 X IO4 M. (A) [NADHIo = 2.56 X M (a); 5.12 X 1W3 M (b); 1.02 X M (c); stirred. (B) [NADHJo= 2.50 X M (a); 5.0 X M (b); 1.0 X M (c); no stirring.
of the reaction solution containing 5.12-20.4 m o l of NADH, 20.2 nmol of MB', and 0.48 Mmol of O2(- 13.3 Mmol of O2in the gas phase above the solution) was inverted several times to stir and to saturate the solution with O2before the absorbance was read. Similar concentrations were used in the experiments shown in Figure 3B, but there was no stirring of the reaction solution in the capped cuvette. The reaction seems to pass through a t least three stages. The first stage is relatively rapid MB+ consumption and the second a very slow decrease in the MB+ concentration. The third stage is characterized by acceleration of the rate of the reduction of MB+. It can be seen in Figure 3B that the second, steady-state phase is shorter than in Figure 3A and depends upon the NADH concentration. Kinetics of the Oxidation of NADH by MB+ in the Absence of 4.The redox reaction was investigated at different pH values and with varying concentrations of NADH and MB+. The reaction shows no induction period or autocatalysis, and exact first-order kinetics are apparent to over 90% completion. Its products are NAD+ and MBH. Good first-order kinetics are obeyed when [MB'] is varied between 4.1 X lod and 1.31 X lo-$ M and [NADH] from 2.42 X lo4 to 3.1 1 X M over the pH range of 7.02-10.56, while keeping at least 22-fold excess of NADH (Figure 4). Therefore, the rate of the reduction of MB+ in the presence of excess NADH and the absence of oxygen may be described by the rate law
s-] for 1 X The value of kob (2.63 X NADH) was found to be independent of hydrogen ion concentration in the range of pH 7.02-10.56 at 25 O C . This suggests that partition between protonation levels of the reactants is negligible over the pM range of the investigation. Changes in buffers (glycine/NaOH or H2P04-/HP042-)or in the buffer concentrations (0.05-0.1 M) have no effect on the rate constant. Hence, there is no evidence for specific or general acid catalysis or for the dependence of rate on proton-transfer reactions.
The Journal of Physical Chemistry, Vol. 95, No. 6,1991
2414
Sevdk and Dunford
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Figure 4. Pseudo-first-orderplots of the oxidation of NADH by MB+ at pH 7.02 (0.1 M phosphate). [MB+]= 9.97 X IOd M; [NADH]: (a) 2.53 X lO-'M, (b) 5.43 X lo-' M, (c) 7.8 X IO-' M, (d) 1.07 X IO-'M, M, (0 2.12 X IO-) M. (e) 1.49 X
,
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+
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-
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2.0
-
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lo3 X [NADH], M Figure 5. Plot of the observed rate constant versus the NADH concentration for the oxidation of NADH by MB+ at pH 8.03 (0.1 M phosphate) (0)and pH 9.0 (0.1 M glycine) ( 0 ) .
The rate constant kobsincreases with the NADH concentration at each pH. A saturation curve is obtained by plotting the kob values against [NADH] (Figure 5). The dependence of kb values on [NADH] can be described by a[NADH] (2) kob = 1 + b[NADH] where a and 6 are parameters discussed later. Equation 2 is verified by a plot of 1/kob versus 1 /[NADH] which from a linear least-squares fit yields a slope (l/a) of 2.80 f 0.001 and an intercept ( 6 / a ) of 99 f 1 from which a and 6 can be calculated. The linearity of Ilkobsversus l/[NADH] is characteristic of a 1:l complex between NADH and MB+, which equilibrates much more rapidly than the hydride transfer between these species. Such a complex may be productive (scheme B) or nonproductive NADH
electron acceptors an upper limit of 1 M-l for the association constant of a productive complex has been indicated." If b[NADH]
