Vol. 183, No. 3, 1992
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March 31, 1992
Pages 983-988
OXIDATION O F D1THIOTHRE1TOL D U R I N G T U R N O V E R O F NITRIC O X I D E REDUCTASE: E V I D E N C E F O R G E N E R A T I O N O F NITROXYL WITH THE ENZYME FROM PARACOCCUS DENI1RIFICANS Tom T u r k l a n d Thomas C. Hollocher Department of Biochemistry, Brandeis University, Waltham, MA 02254 Received January 24, 1992
SUMMARY. The stoichiometric relationship between thiol oxidized and NO reduced was studied for the reaction catalyzed by nitric oxide reductase from Paracoccus denitrificans. The reaction systems consisted of dithiothreitol, ascorbate, phenazine methosulfate, enzyme and NO, or that system m i n u s ascorbate. The mole ratio of thiol groups oxidized to NO reduced was observed to be 2.3 to 1.5 over a range of NO from 0.09 to 0.35 /~mol. A ratio of 1.0 was expected for the simple reduction of NO by 1-electron to N20. The oxidation of additional thiol is attributed to the trapping of nitrosyl hydride (nitroxyl, NO-/NOH) by thiol. ,1992 Ao~d~mioP..... ~no.
Nitric oxide reductase is one of the four reductases of the denitrification pathway in bacteria (1-4), and NO has been shown to be an obligatory intermediate in the pathway (5-8), at least for several bacteria examined. Nitric oxide reductase is associated with the cytoplasmic membrane and has been purified from Pseudomonas stutzeri (1) and Paracoccus denitrificans (3,4) following its solubilization by nonionic detergents. Structurally it appears to be a tight complex of a cytochrome b and a cytochrome c and to contain also some non-heme iron. Except for the fact the nitric oxide reductase reduces NO to N20 with a 2:1 stoichiometry (2,3), little is known about its chemical mechanism. One possibility, which is attractive in its simplicity, is that the enzyme reduces NO by 1-electron to nitrosyl hydride (nitroxyl, NO-/NOH) and then this intermediate converts to NzO as the result of its rapid dimerization and dehydration. The ability of chemically produced nitroxyl to convert to NzO is well established (9-14) and the process appears to be nearly diffusion controlled (15).
In addition, there is evidence that the reduction of NO by ferrous ion in weakly acidic
solutions proceeds via nitroxyl as an intermediate (16,17). Whether nitric oxide reductase reduces NO by an analogous route involving nitroxyl has been the subject of two previous studies (5,18). In an isotope study (18), trioxodinitrate was allowed to decompose into nitroxyl and nitrite in the presence of denitrifying bacteria that were concomitantly reducing [15N]nitrite to NzO.
It was
observed after short incubation times that the 14N/1SN ratio in NzO was much greater than that of Ipresent address: Department of Biology, Biotechnical Faculty, University of Ljubljana, 6100 Ljubljana, Slovenia.
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the nitrite pool and much less than that of the trioxodinitrate pool, a result consistent with dimerization of nitroxyl formed from trioxodinitrate with [15N]nitroxyl formed from [15N]nitrite via the denitrification pathway. The 14N and 15N in N20 were randomized as required for mono-N precursors. However, it could not be excluded that isotopically mixed N20 arose via the reaction of nitroxyl with asNO (1:2) to yield nitrite and NzO (1:1). This reaction has been shown to be significant in the case of nitroxyl produced by pulse radiolysis of NO solutions (19) and by decomposition of trioxodinitrate in presence of NO (15). In a second study (5), an unsuccessful attempt was made to trap extracellular nitroxyl with methemoglobin added to denitrifying bacteria. However, in this and the previous study (18) with intact bacteria, questions concerning the permeability of nitroxyl and its intracellular steady-state levels remained unanswered. In the present study, we attempted to trap nitroxyl arising in the turnover of nitric oxide reductase with a thiol (20). A cell-free system was used in which detergent solubilized nitric oxide reductase served as the enzyme. EXPERIMENTAL PROCEDURES Nitric oxide reductase. Enzyme free of nitrite and nitrous oxide reductase activities was prepared from denitrifying P. denitrificans according to the method of Dermastia et al. (4). "Crude" enzyme was the membrane fraction solubilized between 0.1 and 0.8% octyl glucoside; "partly purified" enzyme was a fraction eluted from the hydroxyapatite column toward the end of the gradient elution step; "purified" enzyme was eluted with 1.5% octyl glucoside in a batch process from hydroxyapatite and had an apparent purity of 90% or greater. Crude, partly purified, and purified enzyme had specific activities of about 0.5, 2-3, and at least 6/xmol NO x min -1 x mg -1, respectively, at p H 6.5 and 25°C in the NO-electrode assay system (4,21). These activities were based simply on the time required to exhaust known quantities of NO and not on maximum rates
(4). Nitric oxide. NO (Matheson) was passed through a 15 x 1.5 cm tube filled with NaOH pellets in order to remove traces of NO z and then used to prepare saturated solutions of NO in anaerobic 20 mM potassium phosphate buffer p H 6.5, 25-30°C. The concentration of NO was taken from tables giving solubility of NO as a function of temperature (22). System to trap nitroxyl with thiol. The system typically contained 10/zl of 1 M sodium ascorbate freshly prepared in water, 10/zl of 0.01 M phenazine methosulfate (PMS) in water, 5 or 10/A of 0.11 M dithiothreitol (DTT) in water, enzyme solution, and sufficient 20 mM potassium phosphate buffer p H 6.5 to bring the final volume to 1 ml. In some experiments, ascorbate was omitted. This mixture was placed in the chamber of a thermostated Clark-type Oz/NO-electrode (Pt/Ag, AgC1, Hansatech model DW-1), and the removal of 02 with use of an argon jet directed at the surface of the solution was followed amperometrically. The nitric oxide reductase reaction was initiated by the injection of 50-200/~1 of buffered NO solution into the anaerobic mixture, and the uptake of NO was followed amperometrically at 25 ° or 30°C. The amount of enzyme added, typically 400/zg of crude, 100 gg of partially purified, or 25/xg of purified enzyme, was sufficient to reduce 0.1 ~mol of NOaq in about 0.5 min and 0.4/~mol in 4-5 min. This lack of proportionality in reaction times was caused by a decrease in the rate of reduction of NO with increasing NO concentrations due to the inhibition of the enzyme by NO (4,6). Immediately after the exhaustion of NO, 100 ~1 of the reaction mixture was mixed with 100/~1 of 6.6 mM 5,5"-dithiobis (2-nitrobenzoic acid)(Ellman's reagent (23)) and then diluted to 5 ml with 0.1 M sodium phosphate buffer p H 8.0. The absorbance at 412 nm due to the thiolate of 2-nitro-5-thiobenzoic acid (e412 = 1.36 x 104 cm -1 x M-1(23)) was determined after 15 min. This 984
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procedure assayed the amount of thiol in D T T after the reduction of NO. The amount of thiol oxidized as a result of the enzymatic reduction of NO was the amount present in an appropriate control system (minus enzyme) minus the amount assayed in the complete system. The compositions of control systems are set forth in connection with Table 1. Controls lacking enzyme were allowed to incubate anaerobically in the electrode chamber for the same lengths of time as were required to exhaust the NO present in the complete enzymatic systems. Immediately thereafter, NO was removed by means of an argon jet and the assay for D T T thiol was carried out on the NO-free mixture. This procedure avoided the oxidation of NO by air to transient products which are known to oxidize thiols (24). Other controls were also incubated for periods of time corresponding to the time needed to exhaust NO enzymatically before D T T was assayed. As reported by Doyle et al. (20), thiols can trap nitroxyl in a two-step reaction (Eq. 1) with production of disulfide and hydroxylamine 1:1. D T T was chosen for the thiol because its redox
RSH + N O H ~ R S N H O H
RSH ~
RSSR + NHzOH
(1)
potential lies well below that of ascorbate at neutrality (25,26). D T T had the potential therefore to reduce dehydroascorbate which was expected to be formed in the initial 1-electron reduction of NO to nitroxyl. Assays. Attempts to detect NH2OH were undertaken with four colorimetric methods: The indooxine assay (27-29), the ferrous chelate method of Belanger et al. (30), the ferricyanide/ phenolphthalein method of Shanine and Mahmoud (31), and the hydroxylamine chelate method of Kavlentis (32). Ascorbate was oxidized after exhaustion or removal of NO by ascorbic acid oxidase (Calbiochem; 200/zmol ascorbate x min -1 x mg -1 at 25°C, p H 5.6). The oxidase (0.1 mg) was added to the reaction mixture in the electrode chamber and stirred under an 0 2 jet. The level of dissolved 02 was monitored and oxidation of ascorbate was judged to be complete when the 02 concentration rose from a steady-state value close to zero to that corresponding to a solution in equilibrium with 0 2 at 1 arm. The time required to oxidize ascorbate was typically 4 min or less. In the presence of PMS, most or all of the remaining D T T was also oxidized by this procedure, presumably via reduction of dehydroascorbate. Protein was assayed by the bicinchoninic acid method (33) using reagents prepared by Pierce.
RESULTS AND DISCUSSION.
The results of a single experiment by way of example are
presented in Table 1. The experiment involved four levels of NO and five kinds of controls, four of which are included in Table 1 (columns A and B and as described in the footnote to Table 1). The observed levels of thiol were sufficiently similar between minus enzyme controls (column A) and minus NO controls (column B) so that the thiol levels for enzymically inactive systems were taken as the average of these two kinds of controls. The control lacking both enzyme and NO provided a value similar to those for the previous two kinds of controls. The similarity among controls lacking enzyme and/or NO suggests that the chemical reduction of NO was relatively unimportant. The control lacking enzyme, NO, ascorbate and PMS (containing only D T r ) gave the highest final levels of thiol.
The lower levels of thiol observed in systems containing
ascorbate/PMS were caused by the presence of some contaminating dehydroascorbate which could oxidize D T T chemically. This ability of DTI" to scavenge dehydroascorbate in presence of PMS simplified the studies, because oxidizing equivalents that may have been distributed between 985
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TABLE 1. Oxidation of thiol groups of DTT during the reduction of nitric oxide in systems containing partially purified nitric oxide reductase, ascorbate, PMS and DTT Initial Amount of NO
control lacking enzyme (A)
Thiol groups remaining in DTT for control lacking average of NO (B) A and B (C)
Complete System (D)
Differential oxidation of thiol groups (C minus D) (E)
/~mol 0.092
2.00
2.01
2.01
1.78
0.23
0.182
1.99
1.91
1.95
1.68
0.27
0.274
1.93
1.86
1.89
1.52
0.37
0.365
1.81
1.92
1.86
1.39
0.47
The procedure was as described under Experimental Procedures and each complete system contained 100/~g of purified enzyme. The control lacking both enzyme and NO had a final content of thiol groups of 1.95 #mol; that lacking enzyme, NO, ascorbate and PMS (containing only DTT), 2.30/zmol. The temperature for equilibration of NO with buffer was 28°C; reaction temperature was 25°C. d e h y d r o a s c o r b a t e a n d D D T disulfide d u r i n g t h e e n z y m a t i c r e a c t i o n a p p e a r e d largely o r e n t i r e l y in D T T disulfide by t h e t i m e t h e thiol assay was u n d e r t a k e n .
T h e fifth c o n t r o l ( d a t a n o t s h o w n )
c o n t a i n e d o n l y N O a n d s e r v e d as a m e a s u r e o f loss o f N O f r o m t h e e l e c t r o d e c h a m b e r , p r e s u m a b l y by w a y largely o f gas p h a s e diffusion t h r o u g h t h e n a r r o w b o r e filling c h a n n e l . T h e loss o f N O was t i m e d e p e n d e n t a n d i n c r e a s e d w i t h t h e initial c o n c e n t r a t i o n o f NOaq. T h i s k i n d o f c o n t r o l a l l o w e d a more
precise estimation of the amount
of NO
t h a t was a c t u a l l y a v a i l a b l e for r e d u c t i o n .
C o r r e c t i o n s f o r t h e a m o u n t o f a v a i l a b l e N O w e r e m a d e in t h e d a t a o f T a b l e 2 b u t n o t o f T a b l e 1. TABLE 2. Oxidation of thiol groups of DTT during the enzymatic reduction of nitric oxide over eight separate experiments Available Presence (+) NO a or absence (-) of 10/zmol of ascorbate
Enzymatic oxidation of thiol groups for systems with crude (C), partially purified (PP), or purified (P) enzyme in parenthesis Exp 1
2
3
/xmol
4
5
Overall Thiol oxidized Average divided by available NO
Ave.
/~mol
0.09
0.23(PP) 0.27(P) 0.24(PP) 0.16(C)
0.22(C) 0.21(C)
0.17(C)
0.18(PP) 0.21±0.04 0.20_+0.04
0.21---0.04
2.3
0.17
+
0.27(PP) 0.32(P) 0.34(PP) 0.22(C)
0.28(C) 0.30(C)
0.19(C)
0.37(PP) 0.29__.0.07 0.29_+0.06
0.29--.0.06
1.7
0.24
+
0.37(PP) 0.41(P) 0.29(PP) 0.24(C)
0.30(C) 0.55(C)
0.35(C)
0.50(PP) 0.39_+0.08 0.36_+0.17
0.38_+0.11
1.6
0.31
+
0.47(PP) 0.32(P) 0.41(PP) 0.37(C)
0.43(C) 0.56(C)
0.43(C)
0.59(PP) 0.45_+0.10 0.45_+0.10
0.45__.0.09
1.5
The procedure was as described under Experimental Procedures. Each experiment involved four different amounts of NO and included those controls described in Table i and the text. The temperature for equilibration of NO with buffer was 28-29°C; reaction temperature was 25° or 30°C. aCorrected for physical loss of NO from the electrode chamber, as described in the text. 986
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Corrections were about 15% when the initial amounts of NO were about 0.4/zmol and when the incubation time was equal to the time required for the enzymatic reduction of the NO.
Such
corrections were negligible when the initial amount of NO was about 0.1/.~mol. As can be seen in Table 1, the enzymatic oxidation of thiol increased with increasing amounts of NO, and the ratio of thiol oxidized to NO added (column EfNO) was greater than one in each case. Table 2 summarizes the results of a group of experiments for which the reducing system was DTT/ascorbate/PMS or DTT/PMS. The fact that little difference was observed between systems with and without ascorbate demonstrated that DTT/PMS can serve as a reductant for nitric oxide reductase. The ratio of thiol oxidized enzymatically to available NO ranged from about 2.3 at the lowest levels of NO used to 1.5 at the highest. Little difference was observed in these parameters between the crude and purified grades of enzyme, so long as the activity levels used were similar and sufficiently great to exhaust the NO within 4-5 min. The 1-electron reduction of NO to nitroxyl would be expected to correspond to the disappearance of one mol of thiol per mol of available NO if the oxidizing equivalents appeared in D T T disulfide at the end of the reaction. If nitroxyl were a freely diffusible intermediate on the path to N20 , it could partition into at least three different reactions: Trapping by D D T (Eq. 1), trapping by NO (Eq. 2) as described by Gr~itzel et al. (19), and dimerization/dehydration (Eq. 3) NO- + 2NO ~ NO 2- + N20
(2)
to yield the normal product, N20. If nitroxyl were trapped quantitatively by DTT, then the 2 N O + 2 H + ~ N20 4- H 2 0 expected overall stoichiometry should be 3 mol of thiol per tool of available NO.
(3) If, instead,
nitroxyl were exclusively trapped by NO or only underwent dimerization/dehydration, then the ratio should be 0.33 or 1, respectively. The observed ratios of 1.5-2.3 are consistent with the trapping of nitroxyl by D T T and therefore with a mechanism of reduction of NO by nitric oxide reductase involving at least some free nitroxyl. The observations that NO is reduced enzymatically to N20 in a 2:1 ratio in absence of thiols (2,3), rather than a 3:1 ratio, suggests that the trapping of nitroxyl by NO (Eq. 2) cannot be very important relative to dimerization/dehydration in the enzymatic reaction. A ratio for N A D H oxidized to NO consumed of 0.5 (instead of something approaching 0.17) with nitric oxide reductase containing membrane vesicles from P. denitrificans (2) also supports this conclusion. Attempts to detect formation of NHzOH in the range of 0.05-0,1/~mol, as expected from the data of Table 2 and Eq. 1, were defeated by interfering materials or an inherent lack of sensitivity, even after the reducing system had been exhaustively oxidized by ascorbic acid oxidase/O2.
In the case of the method of Kavlentis (32), we were unable to reproduce the
published results for assay of hydroxylamine. 987
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Nitroxyl anion, which is isoelectronic with Oz, can apparently exist in two different states of reactivity that have been assigned singlet and triplet electronic states (34). The species formed from the spontaneous decomposition of trioxodinitrate is apparently in the singlet state (34) and can be trapped in part by thiols (20). This implies that the putative nitroxyl generated in the enzymatic reduction of NO is also probably a singlet state species. ACKNOWLEDGMENTS" This work was supported by Grant DCB 88-16273 from the National Science Foundation to T.C.H. and a fellowship to T.T. from the Ministry of Science and Technology of Slovenia. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
Heiss, B., Frunzke, K., and Zumft, W. G. (1989) J. Bacteriol. 171, 3288-3297. Carr, G. J., Page, M. D., and Ferguson, S. J. (1989) Eur. J. Biochem. 179, 683-692. Carr, G. J., and Ferguson, S. J. (1990) Biochem J. 269, 423-429. Dermastia, M., Turk, T., and Hollocher, T. C. (1991) J. Biol. Chem. 266, 10899-10905. Goretski, J., and Hollocher, T. C. (1988) J. Biol. Chem. 263, 2316-2323. Goretski, J., and Hollocher, T. C. (1990) J. Biol. Chem. 265, 889-895. Zafiriou, O. C., Hanley, Q.S., and Snyder, G. (1989) J. Biol. Chem. 264, 5694-5699. Braun, C., and Zumft, W. G. (1991) J. Biol. Chem. 266, 22785-22788. Bonner, F. T., Dzelzkalns, L. S., and Bonucci, J. A. (1978) Inorg. Chem. 17, 2487-2494. Bonner, F. T., and Ravid, B. (1975) Inorg. Chem. 14, 558-563. Hughes, M. N., and Wimbledon, P. E. (1976) J. Chem. Sot., Dalton Trans. 703-707. Bonnet, F. T., and Akhtar, M. (1981) Inorg. Chem. 20, 3155-3160. Angeli, A. (1903) Gazz. Chim. Ital. 33(II), 245-252. Bazylinski, D. A., and Hollocher, T. C. (1985) J. Am. Chem. Soc. 107, 7982-7986. Bazylinski, D. A., and Hollocher, T. C. (1985) Inorg. Chem. 24, 4285-4288. Bonnet, F. T., and Pearsall, K. A. (1982) Inorg. Chem. 24, 1973-1978. Pearsall, K. A., and Bonner, F. T. (1982) Inorg. Chem. 21, 1978-1985. Garber, E. A. E., Wehrli, S., and Hollocher, T. C. (1983) J. Biol. Chem. 258, 3587-3591. Grfitzel, M., Taniguchi, S., and Henglein, A. (1970) Ber. Bunsen-Ges. Phys. Chem. 74, 10031010. Doyle, M. P., Surendra, M. N., Broene, R. D., and Guy, J K. (1988) J. Am. Chem. Soc. 110, 593-599. Ji, X.-B., and Hollocher, T. C. (1988) Appl. Environ. Microbiol. 52, 1791-1794. Lange, N. A. (ed.) (1967) Handbook of Chemistry, 10th edn., McGraw-Hill, N.Y., p. 1101. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. Clancy, R. M., Miyazaki, Y., and Cannon, P. J. (1990). Anal. Biochem. 191, 138-143. Ball, E. G. (1937) J. Bio. Chem. 118, 219-239. Budavari, S. (ed.) (1989) The Merck Index, 11th ed., Merck & Co., Rahway, N.J. Magee, W. E., and Burris, R. H. (1954) Amer. J. Bot. 41, 777-782. Verstraete, W., and Alexander, M. (1972) J. Bacteriol, 110, 955-961. Palling, D. J., and Hollocher, T. C. (1984) J. Org. Chem. 49, 388-390. Belanger, P. M., Grech-Belanger, O., and Blouin, M. (1981) Anal. Chem. 118, 47-52. Shanine, S., and Mahmoud, R. (1978) Microchim. Acta II, 431-434. Kavlentis, E. (1988) Microchem. J. 37, 22-24. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem 150, 76-85. Donald, C. E., Hughes, M. N., Thompson, J. M., and Bonner, F. T. (1986) Inorg. Chem. 25, 2676-2677. 988
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March 31, 1992
Pages 983-988
OXIDATION O F D1THIOTHRE1TOL D U R I N G T U R N O V E R O F NITRIC O X I D E REDUCTASE: E V I D E N C E F O R G E N E R A T I O N O F NITROXYL WITH THE ENZYME FROM PARACOCCUS DENI1RIFICANS Tom T u r k l a n d Thomas C. Hollocher Department of Biochemistry, Brandeis University, Waltham, MA 02254 Received January 24, 1992
SUMMARY. The stoichiometric relationship between thiol oxidized and NO reduced was studied for the reaction catalyzed by nitric oxide reductase from Paracoccus denitrificans. The reaction systems consisted of dithiothreitol, ascorbate, phenazine methosulfate, enzyme and NO, or that system m i n u s ascorbate. The mole ratio of thiol groups oxidized to NO reduced was observed to be 2.3 to 1.5 over a range of NO from 0.09 to 0.35 /~mol. A ratio of 1.0 was expected for the simple reduction of NO by 1-electron to N20. The oxidation of additional thiol is attributed to the trapping of nitrosyl hydride (nitroxyl, NO-/NOH) by thiol. ,1992 Ao~d~mioP..... ~no.
Nitric oxide reductase is one of the four reductases of the denitrification pathway in bacteria (1-4), and NO has been shown to be an obligatory intermediate in the pathway (5-8), at least for several bacteria examined. Nitric oxide reductase is associated with the cytoplasmic membrane and has been purified from Pseudomonas stutzeri (1) and Paracoccus denitrificans (3,4) following its solubilization by nonionic detergents. Structurally it appears to be a tight complex of a cytochrome b and a cytochrome c and to contain also some non-heme iron. Except for the fact the nitric oxide reductase reduces NO to N20 with a 2:1 stoichiometry (2,3), little is known about its chemical mechanism. One possibility, which is attractive in its simplicity, is that the enzyme reduces NO by 1-electron to nitrosyl hydride (nitroxyl, NO-/NOH) and then this intermediate converts to NzO as the result of its rapid dimerization and dehydration. The ability of chemically produced nitroxyl to convert to NzO is well established (9-14) and the process appears to be nearly diffusion controlled (15).
In addition, there is evidence that the reduction of NO by ferrous ion in weakly acidic
solutions proceeds via nitroxyl as an intermediate (16,17). Whether nitric oxide reductase reduces NO by an analogous route involving nitroxyl has been the subject of two previous studies (5,18). In an isotope study (18), trioxodinitrate was allowed to decompose into nitroxyl and nitrite in the presence of denitrifying bacteria that were concomitantly reducing [15N]nitrite to NzO.
It was
observed after short incubation times that the 14N/1SN ratio in NzO was much greater than that of Ipresent address: Department of Biology, Biotechnical Faculty, University of Ljubljana, 6100 Ljubljana, Slovenia.
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the nitrite pool and much less than that of the trioxodinitrate pool, a result consistent with dimerization of nitroxyl formed from trioxodinitrate with [15N]nitroxyl formed from [15N]nitrite via the denitrification pathway. The 14N and 15N in N20 were randomized as required for mono-N precursors. However, it could not be excluded that isotopically mixed N20 arose via the reaction of nitroxyl with asNO (1:2) to yield nitrite and NzO (1:1). This reaction has been shown to be significant in the case of nitroxyl produced by pulse radiolysis of NO solutions (19) and by decomposition of trioxodinitrate in presence of NO (15). In a second study (5), an unsuccessful attempt was made to trap extracellular nitroxyl with methemoglobin added to denitrifying bacteria. However, in this and the previous study (18) with intact bacteria, questions concerning the permeability of nitroxyl and its intracellular steady-state levels remained unanswered. In the present study, we attempted to trap nitroxyl arising in the turnover of nitric oxide reductase with a thiol (20). A cell-free system was used in which detergent solubilized nitric oxide reductase served as the enzyme. EXPERIMENTAL PROCEDURES Nitric oxide reductase. Enzyme free of nitrite and nitrous oxide reductase activities was prepared from denitrifying P. denitrificans according to the method of Dermastia et al. (4). "Crude" enzyme was the membrane fraction solubilized between 0.1 and 0.8% octyl glucoside; "partly purified" enzyme was a fraction eluted from the hydroxyapatite column toward the end of the gradient elution step; "purified" enzyme was eluted with 1.5% octyl glucoside in a batch process from hydroxyapatite and had an apparent purity of 90% or greater. Crude, partly purified, and purified enzyme had specific activities of about 0.5, 2-3, and at least 6/xmol NO x min -1 x mg -1, respectively, at p H 6.5 and 25°C in the NO-electrode assay system (4,21). These activities were based simply on the time required to exhaust known quantities of NO and not on maximum rates
(4). Nitric oxide. NO (Matheson) was passed through a 15 x 1.5 cm tube filled with NaOH pellets in order to remove traces of NO z and then used to prepare saturated solutions of NO in anaerobic 20 mM potassium phosphate buffer p H 6.5, 25-30°C. The concentration of NO was taken from tables giving solubility of NO as a function of temperature (22). System to trap nitroxyl with thiol. The system typically contained 10/zl of 1 M sodium ascorbate freshly prepared in water, 10/zl of 0.01 M phenazine methosulfate (PMS) in water, 5 or 10/A of 0.11 M dithiothreitol (DTT) in water, enzyme solution, and sufficient 20 mM potassium phosphate buffer p H 6.5 to bring the final volume to 1 ml. In some experiments, ascorbate was omitted. This mixture was placed in the chamber of a thermostated Clark-type Oz/NO-electrode (Pt/Ag, AgC1, Hansatech model DW-1), and the removal of 02 with use of an argon jet directed at the surface of the solution was followed amperometrically. The nitric oxide reductase reaction was initiated by the injection of 50-200/~1 of buffered NO solution into the anaerobic mixture, and the uptake of NO was followed amperometrically at 25 ° or 30°C. The amount of enzyme added, typically 400/zg of crude, 100 gg of partially purified, or 25/xg of purified enzyme, was sufficient to reduce 0.1 ~mol of NOaq in about 0.5 min and 0.4/~mol in 4-5 min. This lack of proportionality in reaction times was caused by a decrease in the rate of reduction of NO with increasing NO concentrations due to the inhibition of the enzyme by NO (4,6). Immediately after the exhaustion of NO, 100 ~1 of the reaction mixture was mixed with 100/~1 of 6.6 mM 5,5"-dithiobis (2-nitrobenzoic acid)(Ellman's reagent (23)) and then diluted to 5 ml with 0.1 M sodium phosphate buffer p H 8.0. The absorbance at 412 nm due to the thiolate of 2-nitro-5-thiobenzoic acid (e412 = 1.36 x 104 cm -1 x M-1(23)) was determined after 15 min. This 984
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procedure assayed the amount of thiol in D T T after the reduction of NO. The amount of thiol oxidized as a result of the enzymatic reduction of NO was the amount present in an appropriate control system (minus enzyme) minus the amount assayed in the complete system. The compositions of control systems are set forth in connection with Table 1. Controls lacking enzyme were allowed to incubate anaerobically in the electrode chamber for the same lengths of time as were required to exhaust the NO present in the complete enzymatic systems. Immediately thereafter, NO was removed by means of an argon jet and the assay for D T T thiol was carried out on the NO-free mixture. This procedure avoided the oxidation of NO by air to transient products which are known to oxidize thiols (24). Other controls were also incubated for periods of time corresponding to the time needed to exhaust NO enzymatically before D T T was assayed. As reported by Doyle et al. (20), thiols can trap nitroxyl in a two-step reaction (Eq. 1) with production of disulfide and hydroxylamine 1:1. D T T was chosen for the thiol because its redox
RSH + N O H ~ R S N H O H
RSH ~
RSSR + NHzOH
(1)
potential lies well below that of ascorbate at neutrality (25,26). D T T had the potential therefore to reduce dehydroascorbate which was expected to be formed in the initial 1-electron reduction of NO to nitroxyl. Assays. Attempts to detect NH2OH were undertaken with four colorimetric methods: The indooxine assay (27-29), the ferrous chelate method of Belanger et al. (30), the ferricyanide/ phenolphthalein method of Shanine and Mahmoud (31), and the hydroxylamine chelate method of Kavlentis (32). Ascorbate was oxidized after exhaustion or removal of NO by ascorbic acid oxidase (Calbiochem; 200/zmol ascorbate x min -1 x mg -1 at 25°C, p H 5.6). The oxidase (0.1 mg) was added to the reaction mixture in the electrode chamber and stirred under an 0 2 jet. The level of dissolved 02 was monitored and oxidation of ascorbate was judged to be complete when the 02 concentration rose from a steady-state value close to zero to that corresponding to a solution in equilibrium with 0 2 at 1 arm. The time required to oxidize ascorbate was typically 4 min or less. In the presence of PMS, most or all of the remaining D T T was also oxidized by this procedure, presumably via reduction of dehydroascorbate. Protein was assayed by the bicinchoninic acid method (33) using reagents prepared by Pierce.
RESULTS AND DISCUSSION.
The results of a single experiment by way of example are
presented in Table 1. The experiment involved four levels of NO and five kinds of controls, four of which are included in Table 1 (columns A and B and as described in the footnote to Table 1). The observed levels of thiol were sufficiently similar between minus enzyme controls (column A) and minus NO controls (column B) so that the thiol levels for enzymically inactive systems were taken as the average of these two kinds of controls. The control lacking both enzyme and NO provided a value similar to those for the previous two kinds of controls. The similarity among controls lacking enzyme and/or NO suggests that the chemical reduction of NO was relatively unimportant. The control lacking enzyme, NO, ascorbate and PMS (containing only D T r ) gave the highest final levels of thiol.
The lower levels of thiol observed in systems containing
ascorbate/PMS were caused by the presence of some contaminating dehydroascorbate which could oxidize D T T chemically. This ability of DTI" to scavenge dehydroascorbate in presence of PMS simplified the studies, because oxidizing equivalents that may have been distributed between 985
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TABLE 1. Oxidation of thiol groups of DTT during the reduction of nitric oxide in systems containing partially purified nitric oxide reductase, ascorbate, PMS and DTT Initial Amount of NO
control lacking enzyme (A)
Thiol groups remaining in DTT for control lacking average of NO (B) A and B (C)
Complete System (D)
Differential oxidation of thiol groups (C minus D) (E)
/~mol 0.092
2.00
2.01
2.01
1.78
0.23
0.182
1.99
1.91
1.95
1.68
0.27
0.274
1.93
1.86
1.89
1.52
0.37
0.365
1.81
1.92
1.86
1.39
0.47
The procedure was as described under Experimental Procedures and each complete system contained 100/~g of purified enzyme. The control lacking both enzyme and NO had a final content of thiol groups of 1.95 #mol; that lacking enzyme, NO, ascorbate and PMS (containing only DTT), 2.30/zmol. The temperature for equilibration of NO with buffer was 28°C; reaction temperature was 25°C. d e h y d r o a s c o r b a t e a n d D D T disulfide d u r i n g t h e e n z y m a t i c r e a c t i o n a p p e a r e d largely o r e n t i r e l y in D T T disulfide by t h e t i m e t h e thiol assay was u n d e r t a k e n .
T h e fifth c o n t r o l ( d a t a n o t s h o w n )
c o n t a i n e d o n l y N O a n d s e r v e d as a m e a s u r e o f loss o f N O f r o m t h e e l e c t r o d e c h a m b e r , p r e s u m a b l y by w a y largely o f gas p h a s e diffusion t h r o u g h t h e n a r r o w b o r e filling c h a n n e l . T h e loss o f N O was t i m e d e p e n d e n t a n d i n c r e a s e d w i t h t h e initial c o n c e n t r a t i o n o f NOaq. T h i s k i n d o f c o n t r o l a l l o w e d a more
precise estimation of the amount
of NO
t h a t was a c t u a l l y a v a i l a b l e for r e d u c t i o n .
C o r r e c t i o n s f o r t h e a m o u n t o f a v a i l a b l e N O w e r e m a d e in t h e d a t a o f T a b l e 2 b u t n o t o f T a b l e 1. TABLE 2. Oxidation of thiol groups of DTT during the enzymatic reduction of nitric oxide over eight separate experiments Available Presence (+) NO a or absence (-) of 10/zmol of ascorbate
Enzymatic oxidation of thiol groups for systems with crude (C), partially purified (PP), or purified (P) enzyme in parenthesis Exp 1
2
3
/xmol
4
5
Overall Thiol oxidized Average divided by available NO
Ave.
/~mol
0.09
0.23(PP) 0.27(P) 0.24(PP) 0.16(C)
0.22(C) 0.21(C)
0.17(C)
0.18(PP) 0.21±0.04 0.20_+0.04
0.21---0.04
2.3
0.17
+
0.27(PP) 0.32(P) 0.34(PP) 0.22(C)
0.28(C) 0.30(C)
0.19(C)
0.37(PP) 0.29__.0.07 0.29_+0.06
0.29--.0.06
1.7
0.24
+
0.37(PP) 0.41(P) 0.29(PP) 0.24(C)
0.30(C) 0.55(C)
0.35(C)
0.50(PP) 0.39_+0.08 0.36_+0.17
0.38_+0.11
1.6
0.31
+
0.47(PP) 0.32(P) 0.41(PP) 0.37(C)
0.43(C) 0.56(C)
0.43(C)
0.59(PP) 0.45_+0.10 0.45_+0.10
0.45__.0.09
1.5
The procedure was as described under Experimental Procedures. Each experiment involved four different amounts of NO and included those controls described in Table i and the text. The temperature for equilibration of NO with buffer was 28-29°C; reaction temperature was 25° or 30°C. aCorrected for physical loss of NO from the electrode chamber, as described in the text. 986
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Corrections were about 15% when the initial amounts of NO were about 0.4/zmol and when the incubation time was equal to the time required for the enzymatic reduction of the NO.
Such
corrections were negligible when the initial amount of NO was about 0.1/.~mol. As can be seen in Table 1, the enzymatic oxidation of thiol increased with increasing amounts of NO, and the ratio of thiol oxidized to NO added (column EfNO) was greater than one in each case. Table 2 summarizes the results of a group of experiments for which the reducing system was DTT/ascorbate/PMS or DTT/PMS. The fact that little difference was observed between systems with and without ascorbate demonstrated that DTT/PMS can serve as a reductant for nitric oxide reductase. The ratio of thiol oxidized enzymatically to available NO ranged from about 2.3 at the lowest levels of NO used to 1.5 at the highest. Little difference was observed in these parameters between the crude and purified grades of enzyme, so long as the activity levels used were similar and sufficiently great to exhaust the NO within 4-5 min. The 1-electron reduction of NO to nitroxyl would be expected to correspond to the disappearance of one mol of thiol per mol of available NO if the oxidizing equivalents appeared in D T T disulfide at the end of the reaction. If nitroxyl were a freely diffusible intermediate on the path to N20 , it could partition into at least three different reactions: Trapping by D D T (Eq. 1), trapping by NO (Eq. 2) as described by Gr~itzel et al. (19), and dimerization/dehydration (Eq. 3) NO- + 2NO ~ NO 2- + N20
(2)
to yield the normal product, N20. If nitroxyl were trapped quantitatively by DTT, then the 2 N O + 2 H + ~ N20 4- H 2 0 expected overall stoichiometry should be 3 mol of thiol per tool of available NO.
(3) If, instead,
nitroxyl were exclusively trapped by NO or only underwent dimerization/dehydration, then the ratio should be 0.33 or 1, respectively. The observed ratios of 1.5-2.3 are consistent with the trapping of nitroxyl by D T T and therefore with a mechanism of reduction of NO by nitric oxide reductase involving at least some free nitroxyl. The observations that NO is reduced enzymatically to N20 in a 2:1 ratio in absence of thiols (2,3), rather than a 3:1 ratio, suggests that the trapping of nitroxyl by NO (Eq. 2) cannot be very important relative to dimerization/dehydration in the enzymatic reaction. A ratio for N A D H oxidized to NO consumed of 0.5 (instead of something approaching 0.17) with nitric oxide reductase containing membrane vesicles from P. denitrificans (2) also supports this conclusion. Attempts to detect formation of NHzOH in the range of 0.05-0,1/~mol, as expected from the data of Table 2 and Eq. 1, were defeated by interfering materials or an inherent lack of sensitivity, even after the reducing system had been exhaustively oxidized by ascorbic acid oxidase/O2.
In the case of the method of Kavlentis (32), we were unable to reproduce the
published results for assay of hydroxylamine. 987
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Nitroxyl anion, which is isoelectronic with Oz, can apparently exist in two different states of reactivity that have been assigned singlet and triplet electronic states (34). The species formed from the spontaneous decomposition of trioxodinitrate is apparently in the singlet state (34) and can be trapped in part by thiols (20). This implies that the putative nitroxyl generated in the enzymatic reduction of NO is also probably a singlet state species. ACKNOWLEDGMENTS" This work was supported by Grant DCB 88-16273 from the National Science Foundation to T.C.H. and a fellowship to T.T. from the Ministry of Science and Technology of Slovenia. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
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