6548
are “sociation” rather than “association” constants and the limitations brought forth by Scott8 should be kept in mind when evaluating enthalpies of adduct formation by this procedure. Nevertheless, for systems having enthalpies of adduct formation greater than about 1.5 kcalimol, theseR difficulties should be minimal. The incorrect spectroscopic constants will be obtained only when Y D . ~ / Y D ~ Ais varying or is a constant very much different than unity. T o detect the former complication, we strongly advocate treating the data by a Rose-Drago p r ~ c e d u r e *and ~ ~ looking ~~~ at the intersections as a function of concentration.
H
H
NADH
Ism CH, D
Acknowledgment. We thank the National Science Foundation for its generous support through U. S. NSF GP 31431X.
NAD+
IsHD(I1)
We have previously presented evidence that reduction of flavins by NADH takes place through a preequilibrium complex in which the dihdyronicotinamide does F. L. Slejko, R. S. Drago* not occupy the area adjacent to the 1, 9, and 10 posiDepartment of Chemistry, Unicersity of Illinois tions of the flavin.6 If the present results are interUrbana, INinois 61801 pretable as evidence for transfer of two electrons from Receiced M a y 17, 1971 NADH to the flavin and a proton from the 4 position of the NADH to the 5 position of the flavin, then the geometry of the transition state becomes relatively Demonstration of a Direct Hydrogen Transfer defined. Evidence has been presented for direct hybetween NADH and a Deazaflavin drogen transfer from NADH to substrate aia enzyme bound flavin.’ Sir : In passing it is of interest t o note the general simReductions by NADH have invariably been found ilarities of flavins and I. The second-order rate conto occur by direct transfer of a proton plus two elecstants for the reactions of NADH and NPrNH with trons to the substrate molecule. The mechanism of I [ k , , s . h= ~ ~1.89 M-l min-lat pH 7.62, k 2 , N p r S H = 3.68 the biochemically important reduction of flavins (7,8X l o 2 M-l min-l at pH 7.69 (30”, phosphate buffer dimethyl-10-alkylisoalloxazines) by NADH is uncontaining 5 vol 2 DMF, p = 0.19)] are not too disknown though popular mechanisms reject direct hysimilar from the corresponding rate constants obtained dride transfer and invoke covalent bond formation bewith 3,lO-dimethylisoalloxazine [k2,xADH= 53 M-I tween the dihydronicotinamide and the flavin. The min-’, k2.sprSH = 5.25 X io3 M-l min-1].6 I reacts difficulty in determining whether a direct hydride transwith the component of sulfite buffer to provide fer occurs between NADH and flavin is undoubtedly the 5 adduct,s as previously shown for flavinsSg Upon due to the fact that the protons of the ultimate product acidification of a sulfite-adduct solution in D 2 0 with (1,5-dihydroflavin) are bound to weakly basic nitroDC1, pure I is generated as proven by the nmr spectrum. gens and are, therefore, exchangeable. Since the 5 IsHz regenerates I on reaction with 02,1n as do 1,5-dinitrogen has been shown, r;ia theoretical calculations, hydroflavins,ll and is oxidized by (CH& to yield I. to be the most electrophilic position of the flavin moleThe oxidation of mercaptans by flavins is well estabcule and, therefore, the most likely recipient of a translished6,1zs13 and the reduction of a disulfide by IsHz is ferred hydride ion, we have investigated a compound the retrograde of this reaction. As in the case of flavins, where this nitrogen has been replaced by a carbon. I forms nonfluorescent complexes with tryptophan The reaction of 3,10-dimethyl-5-deazaisoalloxazine and P-resorcylic acid. The 1: 1 complexing constants (I)4 with NADH has been examined in D 2 0 [80 mg of with tryptophan and P-resorcylic acid, determined by I suspended in 5 ml of D 2 0 containing 720 mg of the spectrum of I1 in CDC13 (DMSO-d6): 6 7.6-6.8 (7.6-6.8) [4, ni, Ardisodium salt of NADH was stirred for 3 days in the HI, 3.83 (3.63) [2, S, C(5)-H:], 3.51 (3.30) [3, s, N(lO)-CHd, 3.38 ppm dark (argon atomsphere, 30”); the product (70 mg, (3.16) [3, s, N(3)-CH3]. The proton at position 1 of ISH: shows a 87%) was collected and washed with 2 ml of DzO]. singlet at 6 4.49 ppm in CDCla. (6) T. C. Bruice, L. Main, S . Smith, and P. Y . Bruice, J . Amer. Chew. Except for the deuteron at position 1 the compound Soc., 93,7327 (1971). obtained was indistinguishable by nmr from that ob(7) (a) G . R. Drysdale, Biochim. Biophj,s. Actu, Libr., 8, 159 (1966); (b) P. Strittmatter, ibid., Libr., 8, 325 (1966). tained on reduction of I with dithionite in H20, the (8) 3,10-Dimethyl-5-sulfonate-5-deaza-l,5-dihydroisoalloxazine : A, nmr spectrum” establishing conclusively that the reduc307 nm (E 12,500 iV-1 cm-I) at pH 6.78 ( I O vol % CHaCN, AI = 0.9); tion product was IsHD(I1) of the equation. D M F , p = 0.191, 3 0 “ ; K,, = 3.88 X 102 At4-l at pH 6.93 ( 5 vol (11) T. D. Epley and R. S . Drago, J . Amer. Chem. Soc., 89, 5770 (1967).
( I ) T. C. Bruice and S . J . Benkovic, “Bioorganic Mechanisms,” Vol. 2, W . A . Benjamin, New Y o r k , N. Y.,1966, p 301. ( 2 ) (a) P. Hemmerich, “Flavins and Flavoproteins,” H . Kamin, Ed., University Park Press. Baltimore, Md., 1971, p 103. (b) G. A . Hamilton, Progr. Bioorg. Chenr., 1, 83 (1971). (3) P.-S. Song, S D N (super delocalizability by nucleophile), FOD, and j~~~ calculations, private communication, 1972. (4) Airul. Calcd for C l s H , ~ N 3 0 r :C, 64.72: H. 4.60; h‘, 17.42. Found: C, 64.53; H , 4.55; N, 17.26. ( 5 ) Nmr spectrum of I in CDC13: 6 8.95 [ I , s, C(5)-H], 8.2-7.2 [4. 111, 4r-H], 4.19 [3, s, NIIO)-CHI], 3.47 ppm [3, s, N(3)-CHr]. Nmr
Journal oftlie Americun Chemical Society
1 94:18 /
ki = 2.09 X l o 3 M-’ sec--l at pH 6.89 (5 vol % D M F , p = 0.11, 3 0 ” ; nmr spectrum in D20 6 7.7-6.7 14, m, Ar-HI, 5.13 [ l , s, C(5)-Hl, 3.33 upm .. [6. . s. N(3,10)-CH3], the proton at position 1 shows a singlet at 6 5.4 ppm in H?O. (9) F. Muller and V. Massey, J . Biol. Chem., 244, 4007 (1969). 110) D. E. Edmondson. B. Barman, and G . Tollin, Biochemistr?, 1 1 , 1133 (1972). (1 I ) V. Massey, G. Palmer, and D. Ballou in ref 2a, p 349. (12) I . M. Gascoiene - and G. I
