2111
J . Am. Chem. SOC.1989, 111, 2111-2115
Reduction of Nitrate to Nitrite by Molybdenum-Mediated Atom Transfer: A Nitrate Reductase Analogue Reaction System Julia A. Craig' and R. H. Holm* Contribution from the Department of Chemistry, Harvard University, Cambridge, Massachusetts 021 38. Received July 29, 1988
+
Abstract: The kinetics of the oxygen atom transfer reaction MoIVO(L-NS2)(DMF) NO3- = MoV1O2(L-NS2)+ NO; + DMF was investigated in DMF solution (L-NS2 = 2,6-bis(2,2-diphenyl-2-mercaptoethyl)pyridine(2-)). The reaction is quantitative and well-behaved when conducted in the presence of an excess of nitrate and ca. 1.5 equiv of sulfamic acid, which rapidly scavenges nitrite that otherwise bleaches the Mo chromophores. It is characterized by saturation kinetics in which nitrate reversibly forms a substrate-Mo(IV) complex that generates products in a first-order pathway with kl = (1.49 f 0.05)X s-' at 295.5 K, AH* = 23.7 f 0.6 kcal/mol, and AS* = 8.0 f 2.0eu. The moderate activation entropy suggests that the ground and transition states are structurally similar. The activation enthalpy is indistinguishable from previously reported values for the reductions of S-oxide and N-oxide substrates, which also follow pseudo-first-order kinetics. Inasmuch as the difference in S-0 (Me2SO) and N-O (pyridine N-oxide) bond energies is about 14 kcal/mol, the essentially constant activation enthalpies indicate that the transition state is reached without significant substrate bond weakening. The recently introduced thermodynamic reactivity scale for oxo transfer as applied to substrates with N-0 bonds is discussed. This work contributes the only well-documented reduction of nitrate to nitrite mediated at a Mo(IV) atom and demonstrates that reduction of nitrate by atom transfer is a plausible (but unproven) pathway in the mechanism of action of nitrate reductases.
Among the more familiar molybdoenzymes, often referred to as the molybdenum hydroxylase^,^-^ are the nitrate reductase^.^^^ These enzymes catalyze reaction 1, with NAD(P)H or ferredoxins as common electron donors. The obligatory role of molybdenum
NO3- + 2H+ + 2e-
NO2- + H 2 0 ( E ; = +420 mV)
(1)
in enzymic catalysis has been definitively demonstrated by the reconstitution to full activity of the inactive nitrate reductase from the Neurospora crassa mutant nit-I with molybdenum cofactor released from other molybdoenzymes.8-10 The cofactor is a complex of molybdenum and molybdopterin." A recent EXAFS study of Chlorella vulgaris nitrate reductase suggested the minimal coordination units M o " O ~ ( S R ) ~and , ~ M O ' ~ O ( S R )in~ ,the ~ oxidized and dithionite- and NADP-reduced forms, respe~tive1y.l~ Two of the anionic sulfur ligands presumably derive from the ene-dithiolate chelate group on the side chain of the pterin." By the EXAFS criterion, the molybdenum coordination units in these states of the enzyme resemble those of liver sulfite oxidase." Nitrate reductase from E. coli does not appear to fit into this structural pattern, there being evidence for only one oxo group in the oxidized state and none in the dithionite-reduced form.12 While the somewhat unexpected results for this enzyme may be subject to further examination, the enzymes do differ in being (1) National Science Foundation Predoctoral Fellow, 1986-87. (2) Bray, R. C. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1975; Vol. XII, Part B, Chapter 6 . (3) Bray, R. C. Adu. Enzymol. Relat. Areas Mol. Biol. 1980, 51, 107. (4) Molybdenum and Molybdenum-Containing Enzymes; Coughlan, M. P., Ed.; Pergamon Press: New York, 1980. (5) Molybdenum Enzymes; Spiro, T. G., Ed.; Wiley-Interscience: New York, 1985. (6) Hewitt, E. J.; Notton, B. A. In ref 4, Chapter 8. (7) Adams, M. W. W.; Mortenson, L. E. In ref 5 , Chapter 10. (8) (a) Wahl, R. C.; Hageman, R. V.; Rajagopalan, K. V. Arch. Biochem. Biophys. 1984, 230,264. (b) Kramer, S.; Hageman, R. V.;Rajagopalan, K. V. Arch. Biochem. Biophys. 1984, 233, 821. (9) Hawkes, T. R.; Bray, R. C. Biochem. J . 1984, 219, 481. (IO) Silvestro, A,; Pommier, J.; Giordano, G.Biochim. Biophys. Acta 1986, 872, 243. (1 1) (a) Johnson, J. L.; Rajagopalan, K. V. Proc. Natl. Acad. Sci. U S A . 1982, 79, 6856. (b) Kramer, S . P.; Johnson, J. L.; Ribeiro, A. A.; Millington, D. S.; Rajagopalan, K. V. J . Biol. Chem. 1987, 262, 16357. (12) Cramer, S. P.; Solomonson, L. P.; Adams, M. W. W.; Mortenson, L. E. J . A m . Chem. Soc. 1984, 106, 1467. (13) Cramer, S. P.; Wahl, R.; Rajagopalan, K. V. J . Am. Chem. SOC.1981, 103, 7721.
dissimilatory ( E . coli) and assimilatory (Chlorella). Conceivably, their catalytic sites may not be conserved even though they both catalyze the same reaction. In this laboratory, we have been engaged in the development of analogue reaction systems for m o l y b d o e n ~ y m e s . ~Some ~ of these enzymes we have termed oxotransferases to indicate the possibility that substrate oxidation or reduction may occur without the intervention of any exogenous reactant, as in reaction 2. In MoV102L, + X
+
DMF
x
F?
MoIVOL,
+ XO
e
M o O ~ ( L NS,) -
(2)
xo
MOO( L - N S z ) ( D M F )
(3)
earlier experiments, we have developed the analogue reaction system 3 whose component Mo(IV,VI) complexes have been designed to prevent formation of a p o x 0 Mo(V) dimer and to approach the biological coordination units such as are found in sulfite reductase and Chlorella nitrate reductase. This system is quite effective in oxidizing tertiary phosphines and reducing N-oxides and S - o x i d e ~ , ' ~some - ' ~ of which are enzyme substrates. The purpose of these investigations is to elucidate the kinetics and mechanism of oxidation or reduction of enzyme substrates or pseudosubstrates, with the intention of providing plausible pathways for enzymic reactions. (14) Holm, R. H.; Berg, J. M. Acc. Chem. Res. 1986, 19, 363. (15) Berg, J. M.; Holm, R. H. J . Am. Chem. SOC.1985, 107, 925. (16) Harlan, E. W.; Berg, J. M.; Holm, R. H. J. A m . Chem. Soc. 1986, 108.6992.
0002-7863/89/1511-2111$01.50/00 1989 American Chemical Society
2112 J . A m . Chem. SOC.,Vol. 1 1 1 , No. 6,1989 MoO(L NS2)(OMF) + N O j Mo02(LNS2) + NO;
Craig and Holm
-1, HFQ3H
I I
A
X (nml
Figure 1. Spectral changes in the reaction of 0.82m M MoO(L-NS2)(DMF), 3.0equiv of (n-Bu4N)(N0,), and 1.0 equiv of sulfamic acid in D M F solution at 21 OC. Spectra were recorded every 4 min at a scan rate of 600 nm/min. (These spectra were recorded on a Perkin-Elmer Lambda 4C spectrophotometer.)
Abiological reduction of nitrate by molybdenum has been accomplished with M O ( I I I ) ] ~and *~M ~ o ( V ) . ~ I - ~In~ a number of these systems the initial reaction product is not nitrite but nitrogen dioxide. There is a single, brief claim of the reaction of nitrate with Mo(IV) to afford nitrite and M o ( V I ) ; ~supporting ~ details were not provided. With use of reaction system 3, we have devised an analogue reaction system for nitrate reductase and demonstrated that reduction occurs by atom transfer. The details of this reaction and related observations of nitrate reduction are the subject of this report. The broader matter of oxo transfer reactions mediated at molybdenum and other metal centers is treated elsewhere.30
Experimental Section Preparation 01 Compounds. Mo02(L-NS2)and MoO(L-NS,)(DMF) (L-NS2 = 2,6-bis(2,2-diphenyl-2-mercaptoethyl)pyridine(2-))were synthesized as previously described,” with the minor modification of 10:1 chloroform/DMF (v/v) as the solvent for the reduction of Mo02(L-NS2) with Ph3P. In this medium, MoO(L-NS2)(DMF) precipitates and is obtained in a form more readily soluble in DMF. Kinetics Measurements. All reactions were carried out under strictly anaerobic conditions in solutions of D M F (Burdick & Jackson). The solvent was stored over 3-4 A molecular sieves for several days and degassed immediately before use with two freeze-pump-thaw cycles. (n-Bu4N)(N03) (Fluka) and sulfamic acid (primary standard, G. F. Smith) were used a s received. (Et4N)(N02) was prepared by cation exchange of N a N O z in water on a column of Bio-Rad AG 5 0 W - X 50-100 mesh resin that had been loaded with Et,NOH in methanol and then washed to neutral pH with water. The product was isolated by removal of water from the eluate. Nitrate reduction reactions were monitored spectrophotometrically with use of a Cary 219 or Varian 2390 spectrophotometer and a cell compartment thermostated to f0.5O C . Absorption spectra such as those presented in Figure 1 demonstrate the oxidation of MoO(L-NS,)(DMF) to Mo02(L-NS2) in the presence of nitrate and sulfamic acid. The reaction systems consisted initially of 0.52-0.84 mM MoO(L-NS2)(DMF), 8-1 15 equiv of (n-Bu4N)N03, and 1.5 equiv of sulfamic acid and were examined at four temperatures in the range 281-317 K. (19) Ketchum, P. A.; Taylor, R. C.; Young, D. C. Nature 1976, 259,202. (20) Wieghardt, K.;Woeste, M.; Roy, P. S.; Chaudhuri, P. J . Am. Chem. Sor. 1985, 107, 8276. (21)Guymon, E. P.;Spence, J. T. J . Phys. Chem. 1966, 70, 1964. (22) Garner, C.D.; Hyde, M . R.; Mabbs, F. E.; Routledge, V. I. Nature 1974, 252, 579. (23)Taylor, R. D.; Spence, J. T. Inorg. Chem. 1975, 14, 2815. (24) Garner, C. D.;Hyde, M. R.; Mabbs, F. E.; Routledge, V. I. J. Chem. Soc., Dalton Trans. 1975, 1180. (25) Garner, C.D.; Hyde, M. R.; Mabbs, F. E. Inorg. Chem. 1976, 15, 2327. (26)Durant, R.; Garner, C. D.; Hyde, M . R.; Mabbs, F. E.; Parsons, J . R.; Richens, D. J . Less-Common Mer. 1977, 54, 459. (27) Spence, J. T.; Taylor, R. D. J . Less-Common Met. 1977, 54, 449. (28)Taylor, R. D.;Todd, P. G.; Chasteen, N. D.; Spence, J . T. Inorg. Chem. - _ 1919. 18. 44. (29)Topkhi’J. Inorg. Chim. Acta 1980, 46, L97. (30) Holm, R. H.Chem. Reu. 1987, 87, 1401. (31) Berg, J. M.; Holm, R. H J . A m . Chem. SOC.1985, 107, 917.
Sulfamic acid was introduced to scavenge nitrite, which otherwise bleaches the Mo complexes. The Mo(IV) sulfamic acid solutions were equilibrated in the cell at the temperature of measurement prior to the addition of a concentrated nitrateofilution. Spectra of MoO(L-NS2)(DMF) and Mo02(L-NS2) have been given earlier” and appear in Figure 1. The Mo(IV) spectrum is slightly temperature dependent in that ,,A for the visible band varies from 528 to 536 nm over the temperature range of measurement. The reactions were followed at these maxima. At each of the four temperatures, a t least five runs under pseudo-firstorder conditions were carried out. Observed rate constants were determined from plots of In ( A , - A,) vs time, which were linear for at least 2.5 half-lives. Nonlinearities a t longer times may be due in part to the small quantities of water generated in the scavenging reaction of nitrite with sulfamic acid. In the spectrophotometric monitoring of reactions, perfect isosbestic points could not be obtained in the absence of sulfamic acid. In control experiments at the temperatures and concentrations of the kinetics runs, sulfamic acid altered the apparent extinction coefficients of the Mo(IV) and Mo(VI) complexes by
