J. Phys. Chem. 1983, 87, 3135-3138
The high isotopic selectivity for HF formation in these experiments can be explained by the occurrence of tunneling. Other examples of extreme isotopic selectivity attributed to tunneling phenomena in low-temperature hydrogen-abstraction reactions have recently been reviewed by Le Roy, Murai, and Williams.4o One possible intermediate in the formation of HF in the F + CH3NOz reaction might involve a five-center transition state having a conformation of the type A complex in which the F atom forms a “bridge” between an H atom on the methyl group and the 0 atom to which the F atom is loosely bound. The H-atom “jump” from the CH bond to the stronger HF bond might then occur by a tunneling mechanism.
Conclusions The initial reaction of F atoms with nitromethane involves complexing of the F atom either to the lone-pair electrons on one of the oxygen atoms or to ?r-electronsin an N=O bond. While F-atom addition to CH3NOZ-d,is followed by decomposition of the complex to form HF and (40) R. J. Le Roy, H. Murai, and F. Williams, J.Am. Chem. SOC.,102, 2325 (1980).
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the corresponding nitromethyl radical when n I2, a number of prominent type A absorptions can be attributed to the addition complex with CD3N02. The high degree of isotopic selectivity for the decomposition of the complex to form HF and CHZNO2-d,can be explained by a tunneling mechanism. The threshold for the photodecomposition of this complex into nitromethyl radical and HF or DF lies between 490 and 420 nm. Prominent type B and C absorptions can be assigned to the nitromethyl free radical, which in these experiments is predominantly formed in the solid and, therefore, is hydrogen bonded to HF. The infrared spectrum suggests that this hydrogen bond is relatively strong. The threshold for the photodecomposition of nitromethyl to produce HzCO and NO either directly or by the cage recombination of CH2 and NOz lies between 300 and 280 nm. Acknowledgment. This work was supported in part by the U.S.Army Research Office under Research Proposal 17710-C and by the Office of Naval Research under Contract No. N00014-83-F-0038,NR 659-804. Registry No. CH3N02,75-52-5; CHZNOz,16787-85-2;HF, 766439-3;Hz, 1333-740;F, 14762-948;NF3,7783-542;CHZDNOB, 23171-70-2; CHD2NOz,86013-71-0;CD3N02, 13031-32-8.
One-Electron Transfer Reactions of the Couple NAD/NADH Jan Grodkowskl,’ P. Neta,*2 Brlan W. C a r l s ~ n and , ~ Larry Millers Radiation Laboratory‘ and hpattment of Chemistry, University of Notre Dame, Notre Dame, Indiana 46556; National Bureau of Standards, Washington, D.C. 20234; and Depattment of Chemistry, UniverstYy of Minnesota, Minneapolis, Minnesota 55455 (Received: January 3, 1983)
One-electrontransfer reactions involving the couple NAD./NADH were studied by pulse radiolysis in aqueous solutions. One-electron oxidation of NADH by various phenoxyl radicals and phenothiazine cation radicals was found to take place with rate constants in the range of 105-108 M-’ s-l, depending on the redox potential of the oxidizing species. In all cases, NAD. is formed quantitatively with no indication for the existence of the protonated form (NADH+.). The spectrum of NAD., as well as the rates of oxidation of NADH by phenoxyl and by (chlorpromazine)’. were independent of pH between pH 4.5 and 13.5. Reaction of deuterated NADH indicated only a small kinetic isotope effect. All these findings point to an electron transfer mechanism. On the other hand, attempts to observe the reverse electron transfer, Le., one-electron reduction of NAD. to NADH by radicals such as semiquinones, showed that k was less than 104-106 M-’ s-*, so that it was unobservable. Consequently, it was not possible to achieve equilibrium conditions which would have permitted the direct measurement of the redox potential for NAD./NADH. One-electronreduction of NAD. appears to be an unlikely process.
Introduction The biological importance of the NAD+/NADH couple has stimulated numerous chemical studies on the reversible reaction NAD+ + H+ + 2e F! NADH (1) in the absence of enzymes. The possibility of observation of each of the two one-electron transfer steps independently has attracted special attention.&” NAD+ + e F! NAD. (2) NAD. + e + H+ e NADH (3) (1) University of Notre Dame. On leave of absence from the Institute of Nuclear Research, Warsaw, Poland. (2) University of Notre Dame and National Bureau of Standards. (3) University of Minnesota. (4) The Radiation Laboratory is supported by the Office of Basic This is Document No. Energy Sciences of the Department of Energy. -. NDRL-2411. (5) Land, E. J.; Swallow, A. J. Biochim.Biophys. Acta 1968,162,327. 0022-3654/83/2087-3 135W 1.50/0
k
R
NAD‘
A NAD.
NADH
R = adenosine diphosphoribosyl
The energetics of the first one-electron reduction step has been recently studied by pulse radiolysi~.~J~ The redox (6) Willson. R. L. Chem. Commun. 1970. 1005. (7) Land, E. J.; Swallow, A. J. Biochim.hiophys. Acta 1971,234,34.
(8) Neta, P. Radiat. Res. 1972, 52, 471. (9) Farrington, J. A.; Land, E. J.; Swallow, A. J. Biochim.Biophys. Acta 1980,590, 273. (IO) Anderson, R. F. Biochim. Biophys. Acta 1980,590, 277. (11) Bresnahan, W. T.; Elving, P. J. J.Am. Chem. SOC.1981,103,2379, and references therein.
0 1983 American Chemical Society
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The Journal of Physical Chemistry, Vol. 87, No. 16, 1983
potential for the NAD+/NAD. couple in neutral aqueous solutions has been measured in two laboratories and found to be E,' = -0.92 V'" or -0.94 V9 vs. NHE. Attempts to measure this potential by electrochemical techniques were hampered by the decay of the NAD. radical during the measurements and gave only estimated values." These estimates (ca. -1.0 V vs. SCE) were, however, in reasonable agreement with the pulse radiolysis measurements. The one-electron one-proton redox potential for the second step (reaction 3) can be calculated from the measured values for the equilibria l and 2. The two-electron one-proton redox potential for the NAD+/NADH couple at pH 7 is E: = -0.312 V vs. NHE (as calculated from the value of -0.365 V experimentally measured at pH 8.8).12 Therefore, a potential of E2 = +0.30 V vs. NHE is calculated for the couple NAD./NADH. This value could not be confirmed by electrochemical methods since one-electron oxidation of NADH in those experiments was practically irrever~ib1e.l~ It is, therefore, important to demonstrate the Occurrence of one-electron transfer reactions involving the NAD./ NADH couple and to attempt to obtain an experimental value for the redox potential of this couple by pulse radiolysis. In the present work we use several phenols and anilines, for which the one-electron redox potentials have as well been previously determined by pulse radi~lysis,'~~'~ as several phenothiazines recently ~tudied,'~,'~ in attempts to observe the reaction
S+
+ NADH
S
+ NAD- + H+
(4)
where S+represents the substrate radical cation (e.g., from phenothiazines) or a substrate neutral radical (e.g., PhO from phenols).
Experimental Section The compounds used were of the purest grade commercially available and were used without further purification. Nicotinamide adenine dinucleotide in ita oxidized (NAD+)and reduced (NADH) forms was obtained from Sigma. The latter was also obtained from P. L. Biochemicals. NADH with one deuterium at the 4 position was synthesized by the method of Brown and Fisherl8 and isolated as the barium salt. Its purity was confirmed by NMR. p-Methoxyphenol, hydroquinone, and p-aminophenol were from Aldrich. Catechol and N,N,N',N'tetramethyl-p-phenylenediamine(TMPD) were from Eastman. Pyrogallol and sodium formate were from Baker, resorcinol and phenol were from Mallinckrodt, ethylene glycol, 2-propanol, and KBr were from Fisher, 1,2,4-trihydroxybenzene was from Pfaltz and Bauer, and ascorbic acid was from Fluka. Chlorpromazinewas purchased from Sigma and promazine and promethazine were kindly supplied by Dr. E. Pelizzetti. Nitrous oxide was obtained from Linde. Water was purified by a Millipore Milli-Q system. Solutions were saturated with N20and passed through the irradiation cell in a flow system at room temperature, 21 f 1"C. Kinetic spectrophotometric experiments were carried out with the computer-controlled pulse radiolysis apparatus described previo~s1y.l~The linear accelerator (12)Srinivaean, R.;Medary, R. T.; Fisher, H. F.; Noms, D. J.; Stewart, R.J . Am. Chem. SOC.1982,104,807.
(13)Moiroux, J.; Elving, P. J. J. Am. Chem. SOC.1980,102, 6533. (14)Steenken, S.;Neta, P. J.Phys. Chem. 1979,83, 1134. (15)Steenken, S.;Neta, P. J . Phys. Chem. 1982,86, 3661. (16)Pelizzetti, E.; Meisel, D.; Mulac, W. A.; Neta, P. J.Am. Chem. SOC. 1979,101,6954. (17)Pelizzetti, E.; Mentasti, E. Inorg. Chem. 1979,18,583. (18)Brown, A.;Fisher, H. F. J. Am. Chem. SOC.1976,98, 5682.
supplied 10-ns 8-MeV electron pulses which deposited energy in the solution to produce 3-4 pM total radical concentration. The wavelengths for kinetic measurements were chosen according to the spectra of the investigated radicals as reported in the l i t e r a t ~ r e . ~ ? ~ J ~ - ' ~ The radiolysis of aqueous solutions produces e@-,H, and OH radicals. N20 converts e, into OH, which then reacts with the organic substrate. Alternatively, OH was converted into another oxidant, e.g., by reacting with Br- to form Brz-., which in turn reacted with the organic substrate.
Results and Discussion In order to examine the occurrence of the equilibrium reaction 4 it is advantageous to attempt to measure the kinetics for both forward and reverse reactions under favorable conditions, Le., to observe one-electron oxidation of NADH and one-electron reduction of NAD., with various reagents. One-Electron Oxidation of NADH. It has already been demonstrated7that NADH is oxidized by the Br,-- radical rapidly and quantitatively to form NAD-. Although oneelectron oxidation of NADH may yield initially the cation radical NADH+., no evidence for the existence of this cation radical in neutral aqueous solutions was found; NAD. is produced by the oxidation without any apparent delay (
