Vol.
175,
March
No.
29,
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1991
CATALYSIS
OF NITROSYL TRANSFER BY DENITRIFYING IS FACILITATED BY NITRIC OXIDE
901-905
BACTERIA
Joanne Goretski and Thomas C. Hollocher Department Received
January
24,
of Biochemistry,
Brandeis University, Waltham,
MA 02254
1991
SUMMARY. Two denitrifying bacteria, Pseudomonasstatzeri and Achromobacter cycloclastes,were incubated with Nal’NO, and NaN, under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and coppercontaining nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14P15N20,whereas denitrification leads to lSN,O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of “NO. Absorption of NO from the gas phase by acidic CrSO, in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of 0 1991 Academic denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO. Press, Inc. It was shown that denitrifying
bacteria (1) and the heme-containing
nitrite
reductase of
Pseudomonasaerugzkosa(2) could catalyze the transfer under anaerobic conditions of the nitrosyl
group (NO+) from [“Nlnitrite
to N-nucleophiles
transfer to 0 of water resulted in a nitrite/water
with formation
of r4@N20 or r4j1’N2. Nitrosyl
-I*0 exchange and incorporation
of 180 into N,O.
Exchange of 180 appeared to involve an enzyme-bound species that could partition ciation to form [“Olnitrite
and reduction
to yield N,180.
Later studies with cells and cell-free
extracts from P. stutzeri and A. cycloclastes(3-6) revealed several additional system. First, higher ratios of [“N]nitrite products. “N,O of denitrification
might arise either by the reduction of [‘5N]nitrite or by a transfer of “NO+ intermediate
characteristics of the
to azide increased the ratio of lSN,O to r4~r5N20 as to [“N]nitrite-N
via 15N0 in the classical pathway with subsequent reduction
dehydration steps (7). The observation of what might be competition a nitrosyl-donating
between disso-
supported the latter possibility.
and
between nitrite and azide for
Second, the 180 content of “N,O
could be lower than (rather than nearly equal to (1)) that of 14,1sN20when the reactions were run in H,180 with azide as the nitrosyl acceptor. This result was consistent with either dilution of “0 by 160 in the second nitrite molecule or O-exchange by an intermediate
in the nitrosation
Third, increasing concentrations of nitrite tended to suppress incorporation competition
reaction.
of 180 into N,O. These
and exchange data were used to argue (3-6) that the mechanism
of denitrification
involves the obligatory transfer of nitrosyl from one nitrite molecule to a second nitrite molecule
901
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and that the critical N-N bond forming step occurs at the N3+ redox level. On the other hand, a considerable body of enzymic, genetic and analytical data (8-24) supports the view that the major pathway of denitrification
involves NO as an obligatory intermediate,
so that N-N bond formation
first occurs at the N2+ or Nt state through the action of nitric oxide reductase. In considering this paradox, it occurred to us that most, if not all, of the in vivo and in vitro systems used to demonstrate
nitrosyl transfer reactions were inhibited
in or lacked nitric oxide
reductase activity and therefore accumulated substantial amounts of NO. Some indication circumstance had been previously reported (1). Normally, during denitrification
high concentrations
predispose it to nitrosation interesting commitment
of NO might perturb the system and in particular
reactions. The two bacteria used, P. stutzeti and A. cycloclastes, were
to compare, inasmuch as their respiratory nitrite
cytochrome c,d, and Cu types, respectively. important
of NO,,
in vivo is very low (l-60 nM)( 10, 11). We considered therefore whether the
presence of abnormally particularly
the steady-state concentration
of this
reductases are of the
They thus afford different classes of enzyme at the
step of denitrification.
METHODS. P. stutzeri JM300 (from J. L. Ingraham, University of California, Davis) and A. ATCC 21921 were grown anaerobically at 30-32” C in medium consisting of 6 g of yeast extract and 3 g of peptone per liter of 16 mM potassium phosphate buffer pH 7.4 and containing 10 mM KNO, as oxidant. Maintenance of cultures and harvesting procedures were as described previously (9, 25). Bacteria were suspended at 1 mg of cell protein x ml-’ in cold fresh medium lacking nitrate and used within 4 h. Na”NO, (99 atom %) was from Cambridge Isotope Laboratories, nutrient powders were from Difco and Cr metal (99.995% pure) was from Fluka Chemie, A. G. Acidic CrSO, (used to trap NO) was prepared anaerobically by the reaction of 0.5 g of Cr with 1 ml of cont. H,SO, in 4 ml of water at 90°C. A portion (0.3 ml) of the resulting bright blue solution was transferred to the sidewell, which contained a magnetic flea, of a 9-ml reaction vial. The preparation of CrSO, made use of a Coy anaerobic chamber. All rubber septa were exposed to NO before use (9). Cell protein was estimated by the method of Lowry et al. (26) that uses NaOH to solubilize cells. Nitrite was determined by a calorimetric method (27). The isotopic composition of NOs and N,O, in the headspace of vigorously stirred reaction vials incubated at 25°C was determined with use of a Hewlett-Packard 5992A GC-MS as previously described (9, 28). For these experiments, 1 ml of bacterial suspension was placed in a 9-ml vial under He. Additions were, variously, 10 mM NalSN02, 50 mM NaN, and acetylene to 0.1 atm. Acetylene was used to inhibit nitrous oxide reductase (29,30) when azide was absent. Azide alone completely inhibited this enzyme. Because acetylene reacted with CrSO, in the sidewell, it was omitted whenever CrSO, was used. cyclochtes
RESULTS
AND DISCUSSION.
In systems which lacked azide but contained acetylene to
inhibit N,O reduction, the rate of reduction of [“Nlnitrite
was 0.33 and 0.25 pmol x min -’ x mg-’
of cell protein for P. s&hen’ and A. cycloclastes, respectively. steady-state levels of total NO (NO, plus NO,)
NO failed to accumulate
and the
were 0.01-0.02 pmol (8 ml headspace, 1.1-1.3 ml 902
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3, 1991
BIOCHEMICAL
aq vol) from data at m/e = 31. equilibrium
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These levels corresponded to 28-56 patm for NO, and an
of 56-111 nM for NO,,,
Both the rates of denitrification
and the steady-
state NO,, concentrations were in reasonable accord with published values for denitrifying bacteria (1, S-11, 31, 32). The signal at m/e = 45 ( 14V15N20)was 0.012-0.014 of the signal at m/e = 46 (i5Nz0).
A value of 0.02 was expected for 99% [rSN]nitrite.
These results indicate that [“Nlnitrite
was the only source of N for denitrification. The presence of 50 mM azide caused a roughly 3-fold decrease in the rate of processing of [“Nlnitrite
and the production
14,1sN,0/‘5N,0
of substantial
amounts of 14,15N20 (Table
1).
The overall
ratio was 0.26 and 0.76, respectively, for P. stitzeti and A. qcloclustes.
bacteria, “NO accumulated due to inhibition
of the NO-reducing
With both
system by azide (9). Introduction
of C&O, into a stirred sidewell had two effects (Table 1). First lSNO failed to accumulate and was held to low levels.
reaction leading to 14*15N20 was largely abolished
Second, the nitrosation
relative to the reactions leading to “N,O. depends on NO.
It would appear that the catalysis of nitrosyl transfer
In the absence of azide, CrSO, in the sidewell seemed to have no effect on the
kinetics of denitrification. One explanation
CrSO, also did not reduce N,O.
for the results is that NO may act on the nitrite reductase system by mass
action to shift the steady-state distribution
of enzyme species to favor the nitrosyl donor species
(Eq- 1).
E-Fe2+
+
NO;
$
E-Fe2+
l
NO;
+
E-Fe2’
l
NO+ +
E-Fe3’
l
NO e
E-Fe3+ + NO
(1)
The NO-bound
species is thought to be the nitrosyl donor and an analogous eq. is assumed to apply
with Cu-nitrite
reductase.
Nitrite/water-
(l-5) and NO/water-“0
presumed to occur at the dehydration/hydration
(33) exchange reactions are
step. The initial product of nitrosyl transfer from
nitrite to nitrite may be N,O,, perhaps bound to enzyme (7). If this dimeric product were reducible to N,O, it would provide a pathway to “N,O as an intermediate.
separate from the classical one which involves NO
Eq. 1 might also explain how the Cu-nitrite
reductase of A. cydoclastes, that
normally reduces nitrite to NO and is inert towards NO, can nevertheless reduce nitrite to N,O in the presence of NO (33). enzyme of denitrifying
This capability
has not yet been reported for the heme-containing
bacteria.
Because nitrosation reactions (and perhaps the putative dimeric pathway to N,O) may depend on or be controlled by NO, it is possible that the dimeric pathway may serve as a metabolic escape route for reduction of nitrite when nitric oxide reductase is inhibited. In the course of this work, several different NO traps were tested. Some, such as alkaline sulfite and concentrated solutions of hemoglobin,
were kinetically 903
slow. Acidic permanganate
was
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TABLE 1. Effect of absorption of “NO on the nitrosation of azide by [“N]Nitrite as catalyzed by P. sr~tren’ and A. cycZocZu.rfeP Organism
P. stutzeri
CrSO, trap, present or absent
absent
present
A. cycloclasfes
absent
present
Incubation time
“NO
Accumulated 14s15N20 “N20 pm01
14~1sN,0/‘5N,0 increoverall mental
pm01
mill
pm01
2 12 22 32 42 52 62 72
0.20 0 1.6 -0.01 2.4 0.05 2.9 0.11 3.1 0.18 3.3 0.26 3.5 0.35 3.6 0.47
2 12 22 32 42 52 62 72
0.10 -0.005 0.06 -0.01 0.03 0.15 ,O.Ol 0.019 0 0.029 0 0.040 0 0.051
0.18 0.43 0.67 1.03 1.56 2.35 3.35 4.23
0 -2.0 -2.1 1.4 0.8 1.3 1.1 1.2
2 11 20 29 38 47 56 65
0.36 2.06 3.31 3.74 4.25 4.71 5.09 5.48
0.072 0.19 0.25 0.31 0.36 0.41 0.46 0.51
0 30 130 151 148 117 61 50
2 11 20 29 38 47 56
0.13 0 0.10 -0.007 0.09 0.014 0.07 0.022 0.07 0.028 0.07 0.032 0.05 0.035
0.04
0 -8.8 12 15 12 8.2 4.8
0
0
0.036 0.11 0.20 0.28 0.33 0.36 0.38
%
%
0
0.09
0.37 0.59 0.82 1.07 1.33 1.57 1.81
-11 18 26 28 31 37 48
0.12 0.18 0.23 0.28 0.33 0.40
26
1.2
76
8.9
“The systems were asdescribedin METHODS andcontained1 mgof cell protein, 10mM NanNO, and 50 mM NaN,. Total volume was 1.1-1.3ml and headspacevolume wasabout 8 ml.
kinetically effective in NO uptake but produced
a nitrosating
species (presumably
N,O,)
in
sufficient amounts so as to make it useless for the experiments at hand. CrSO,, because of its well known ability to reduce NO to ammonia,
avoided this problem.
None of the traps tested reacted
with N,O, in so far as we could determine.
This work was supported by grant DCB 88-16273 from the National 904
Science Foundation.
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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.
Garber, E. A. E., and Hollocher, T. C. (1982) J. Biol. Chem. 257, 8091-8097. Kim, C.-H., and Hollocher, T. C. (1984) J. Biol. Chem. 259, 2092-2099. Aerssens, E., Tiedji, J. M., and Averill, B. A. (1986) J. Biol. Chem. 261, 9652-9656. Weeg-Aerssens, E., Tiedji, J. M., and Averill, B. A. (1987) J. Am. Chem. Sot. 109, 7214-7215. Weeg-Aerssens, E., Tiedji, J. M., and Averill, B. A. (1988) J. Am. Chem. SOC.~, 6851-6856. Hulse, C. L., and Averill, B. A. (1989) J. Am. Chem. Sot. 111, 2322-2323. Averill, B. A., and Tiedji, J. M. (1982) FEBS Lett. 138, 8-11. 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. Goretski, J., Zafiriou, 0. C., and Hollocher, T. C. (1990) J. Biol. Chem. 2& 11535-11538. Zafiriou, 0. C., Hanley, Q. S., and Snyder, G. (1989) J. Biol. Chem. 264, 5694-5699. Heiss, B., Frunzke, K., and Zumft, W. G. (1989) J. Bacterial. 171, 3288-3297. Dermastia, M., Turk, T., and Hollocher, T. C. (1991) J. Biol. Chem. 266, submitted. Carr, G. J., and Ferguson, S. J. (1990) Biochem. J. 269, 423-429. Zumft, W. G., Dohler, K., Korner, H., Liichelt, S., Viebrock, A., and Fnmzke, K. (1988) Arch. Microbial. 149, 491-498. Shapleigh, J. P., and Payne, W. J. (1985) FEMS Microbial. L&t. 26, 275-279. Shapleigh, J. P., Davies, K. J. P., and Payne, W. J. (1987) Biochim. Biophys. Actam, 334-340. Shapleigh, J. P., and Payne, W. J. (1985) J. Bacterial. 163, 837-840. Garber, E. A. E., Castignetti, D., and Hollocher, T. C. (1982) Biochem. Biophys. Res. Commun. 107, 1504-1507. Mancinelli, R. L., Cronin, S., and Hochstein, L. I. (1986) Arch. Microbial. 145, 202-208. Kim, C.-H., and Hollocher, T. C. (1983) J. Biol. Chem. 258, 4861-4863. Hulse, C. L., Tiedje, J. M. and Averill, B. A. (1988) Anal. Biochem. 172, 420-426. Silverstrini, M. C., Tordi, M. G., Musci, G., and Brunori, M. (1990) J. Biol. Chem. 265, 11783-11787. Firestone, M. K., Firestone, R. B., and Tiedji, J. M. (1979) Biochem. Biophys. Res. Commun. !X, 10-16. Garber, E. A. E., and Hollocher, T. C. (1981) J. Biol. Chem. B, 5459-5465. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Snell, F. D., and Snell, C. T., (1949) Calorimetric Method of Analysis, Vol. 2, 3rd Edn., pp. 804-805, D. Van Nostrand Co., New York. Bazylinski, D. A., SooHoo, C. K., and Hollocher, T. C. (1986) Appl. Environ. Microbial. 51, 1239-1246. Balderston, W. L., Sherr, B., and Payne, W. J. (1976) Appl. Environ. Microbial. 31, 504-508. Yoshinari, T., and Knowles, R. (1976) Biochem. Biophys. Res. Comrnun. 69, 705-710. St. John, R. T., and Hollocher, T. C. (1977) J. Biol. Chem. 252, 212-218. Walter, B., Sidransky, E., Kristjansson, J. K., and Hollocher, T. C. (1978) Biochemistry 17, 3039-3045. Averill, B. A., and Tiedji, J. M. (1990) Abstract INOR 103, Symp. on Inorg. and Biol. Chem. of Nitrogen, annu. meeting, Am. Chem. Sot., Washington, D.C., August 26-31.
905
175,
March
No.
29,
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1991
CATALYSIS
OF NITROSYL TRANSFER BY DENITRIFYING IS FACILITATED BY NITRIC OXIDE
901-905
BACTERIA
Joanne Goretski and Thomas C. Hollocher Department Received
January
24,
of Biochemistry,
Brandeis University, Waltham,
MA 02254
1991
SUMMARY. Two denitrifying bacteria, Pseudomonasstatzeri and Achromobacter cycloclastes,were incubated with Nal’NO, and NaN, under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and coppercontaining nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14P15N20,whereas denitrification leads to lSN,O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of “NO. Absorption of NO from the gas phase by acidic CrSO, in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of 0 1991 Academic denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO. Press, Inc. It was shown that denitrifying
bacteria (1) and the heme-containing
nitrite
reductase of
Pseudomonasaerugzkosa(2) could catalyze the transfer under anaerobic conditions of the nitrosyl
group (NO+) from [“Nlnitrite
to N-nucleophiles
transfer to 0 of water resulted in a nitrite/water
with formation
of r4@N20 or r4j1’N2. Nitrosyl
-I*0 exchange and incorporation
of 180 into N,O.
Exchange of 180 appeared to involve an enzyme-bound species that could partition ciation to form [“Olnitrite
and reduction
to yield N,180.
Later studies with cells and cell-free
extracts from P. stutzeri and A. cycloclastes(3-6) revealed several additional system. First, higher ratios of [“N]nitrite products. “N,O of denitrification
might arise either by the reduction of [‘5N]nitrite or by a transfer of “NO+ intermediate
characteristics of the
to azide increased the ratio of lSN,O to r4~r5N20 as to [“N]nitrite-N
via 15N0 in the classical pathway with subsequent reduction
dehydration steps (7). The observation of what might be competition a nitrosyl-donating
between disso-
supported the latter possibility.
and
between nitrite and azide for
Second, the 180 content of “N,O
could be lower than (rather than nearly equal to (1)) that of 14,1sN20when the reactions were run in H,180 with azide as the nitrosyl acceptor. This result was consistent with either dilution of “0 by 160 in the second nitrite molecule or O-exchange by an intermediate
in the nitrosation
Third, increasing concentrations of nitrite tended to suppress incorporation competition
reaction.
of 180 into N,O. These
and exchange data were used to argue (3-6) that the mechanism
of denitrification
involves the obligatory transfer of nitrosyl from one nitrite molecule to a second nitrite molecule
901
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and that the critical N-N bond forming step occurs at the N3+ redox level. On the other hand, a considerable body of enzymic, genetic and analytical data (8-24) supports the view that the major pathway of denitrification
involves NO as an obligatory intermediate,
so that N-N bond formation
first occurs at the N2+ or Nt state through the action of nitric oxide reductase. In considering this paradox, it occurred to us that most, if not all, of the in vivo and in vitro systems used to demonstrate
nitrosyl transfer reactions were inhibited
in or lacked nitric oxide
reductase activity and therefore accumulated substantial amounts of NO. Some indication circumstance had been previously reported (1). Normally, during denitrification
high concentrations
predispose it to nitrosation interesting commitment
of NO might perturb the system and in particular
reactions. The two bacteria used, P. stutzeti and A. cycloclastes, were
to compare, inasmuch as their respiratory nitrite
cytochrome c,d, and Cu types, respectively. important
of NO,,
in vivo is very low (l-60 nM)( 10, 11). We considered therefore whether the
presence of abnormally particularly
the steady-state concentration
of this
reductases are of the
They thus afford different classes of enzyme at the
step of denitrification.
METHODS. P. stutzeri JM300 (from J. L. Ingraham, University of California, Davis) and A. ATCC 21921 were grown anaerobically at 30-32” C in medium consisting of 6 g of yeast extract and 3 g of peptone per liter of 16 mM potassium phosphate buffer pH 7.4 and containing 10 mM KNO, as oxidant. Maintenance of cultures and harvesting procedures were as described previously (9, 25). Bacteria were suspended at 1 mg of cell protein x ml-’ in cold fresh medium lacking nitrate and used within 4 h. Na”NO, (99 atom %) was from Cambridge Isotope Laboratories, nutrient powders were from Difco and Cr metal (99.995% pure) was from Fluka Chemie, A. G. Acidic CrSO, (used to trap NO) was prepared anaerobically by the reaction of 0.5 g of Cr with 1 ml of cont. H,SO, in 4 ml of water at 90°C. A portion (0.3 ml) of the resulting bright blue solution was transferred to the sidewell, which contained a magnetic flea, of a 9-ml reaction vial. The preparation of CrSO, made use of a Coy anaerobic chamber. All rubber septa were exposed to NO before use (9). Cell protein was estimated by the method of Lowry et al. (26) that uses NaOH to solubilize cells. Nitrite was determined by a calorimetric method (27). The isotopic composition of NOs and N,O, in the headspace of vigorously stirred reaction vials incubated at 25°C was determined with use of a Hewlett-Packard 5992A GC-MS as previously described (9, 28). For these experiments, 1 ml of bacterial suspension was placed in a 9-ml vial under He. Additions were, variously, 10 mM NalSN02, 50 mM NaN, and acetylene to 0.1 atm. Acetylene was used to inhibit nitrous oxide reductase (29,30) when azide was absent. Azide alone completely inhibited this enzyme. Because acetylene reacted with CrSO, in the sidewell, it was omitted whenever CrSO, was used. cyclochtes
RESULTS
AND DISCUSSION.
In systems which lacked azide but contained acetylene to
inhibit N,O reduction, the rate of reduction of [“Nlnitrite
was 0.33 and 0.25 pmol x min -’ x mg-’
of cell protein for P. s&hen’ and A. cycloclastes, respectively. steady-state levels of total NO (NO, plus NO,)
NO failed to accumulate
and the
were 0.01-0.02 pmol (8 ml headspace, 1.1-1.3 ml 902
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BIOCHEMICAL
aq vol) from data at m/e = 31. equilibrium
concentration
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These levels corresponded to 28-56 patm for NO, and an
of 56-111 nM for NO,,,
Both the rates of denitrification
and the steady-
state NO,, concentrations were in reasonable accord with published values for denitrifying bacteria (1, S-11, 31, 32). The signal at m/e = 45 ( 14V15N20)was 0.012-0.014 of the signal at m/e = 46 (i5Nz0).
A value of 0.02 was expected for 99% [rSN]nitrite.
These results indicate that [“Nlnitrite
was the only source of N for denitrification. The presence of 50 mM azide caused a roughly 3-fold decrease in the rate of processing of [“Nlnitrite
and the production
14,1sN,0/‘5N,0
of substantial
amounts of 14,15N20 (Table
1).
The overall
ratio was 0.26 and 0.76, respectively, for P. stitzeti and A. qcloclustes.
bacteria, “NO accumulated due to inhibition
of the NO-reducing
With both
system by azide (9). Introduction
of C&O, into a stirred sidewell had two effects (Table 1). First lSNO failed to accumulate and was held to low levels.
reaction leading to 14*15N20 was largely abolished
Second, the nitrosation
relative to the reactions leading to “N,O. depends on NO.
It would appear that the catalysis of nitrosyl transfer
In the absence of azide, CrSO, in the sidewell seemed to have no effect on the
kinetics of denitrification. One explanation
CrSO, also did not reduce N,O.
for the results is that NO may act on the nitrite reductase system by mass
action to shift the steady-state distribution
of enzyme species to favor the nitrosyl donor species
(Eq- 1).
E-Fe2+
+
NO;
$
E-Fe2+
l
NO;
+
E-Fe2’
l
NO+ +
E-Fe3’
l
NO e
E-Fe3+ + NO
(1)
The NO-bound
species is thought to be the nitrosyl donor and an analogous eq. is assumed to apply
with Cu-nitrite
reductase.
Nitrite/water-
(l-5) and NO/water-“0
presumed to occur at the dehydration/hydration
(33) exchange reactions are
step. The initial product of nitrosyl transfer from
nitrite to nitrite may be N,O,, perhaps bound to enzyme (7). If this dimeric product were reducible to N,O, it would provide a pathway to “N,O as an intermediate.
separate from the classical one which involves NO
Eq. 1 might also explain how the Cu-nitrite
reductase of A. cydoclastes, that
normally reduces nitrite to NO and is inert towards NO, can nevertheless reduce nitrite to N,O in the presence of NO (33). enzyme of denitrifying
This capability
has not yet been reported for the heme-containing
bacteria.
Because nitrosation reactions (and perhaps the putative dimeric pathway to N,O) may depend on or be controlled by NO, it is possible that the dimeric pathway may serve as a metabolic escape route for reduction of nitrite when nitric oxide reductase is inhibited. In the course of this work, several different NO traps were tested. Some, such as alkaline sulfite and concentrated solutions of hemoglobin,
were kinetically 903
slow. Acidic permanganate
was
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TABLE 1. Effect of absorption of “NO on the nitrosation of azide by [“N]Nitrite as catalyzed by P. sr~tren’ and A. cycZocZu.rfeP Organism
P. stutzeri
CrSO, trap, present or absent
absent
present
A. cycloclasfes
absent
present
Incubation time
“NO
Accumulated 14s15N20 “N20 pm01
14~1sN,0/‘5N,0 increoverall mental
pm01
mill
pm01
2 12 22 32 42 52 62 72
0.20 0 1.6 -0.01 2.4 0.05 2.9 0.11 3.1 0.18 3.3 0.26 3.5 0.35 3.6 0.47
2 12 22 32 42 52 62 72
0.10 -0.005 0.06 -0.01 0.03 0.15 ,O.Ol 0.019 0 0.029 0 0.040 0 0.051
0.18 0.43 0.67 1.03 1.56 2.35 3.35 4.23
0 -2.0 -2.1 1.4 0.8 1.3 1.1 1.2
2 11 20 29 38 47 56 65
0.36 2.06 3.31 3.74 4.25 4.71 5.09 5.48
0.072 0.19 0.25 0.31 0.36 0.41 0.46 0.51
0 30 130 151 148 117 61 50
2 11 20 29 38 47 56
0.13 0 0.10 -0.007 0.09 0.014 0.07 0.022 0.07 0.028 0.07 0.032 0.05 0.035
0.04
0 -8.8 12 15 12 8.2 4.8
0
0
0.036 0.11 0.20 0.28 0.33 0.36 0.38
%
%
0
0.09
0.37 0.59 0.82 1.07 1.33 1.57 1.81
-11 18 26 28 31 37 48
0.12 0.18 0.23 0.28 0.33 0.40
26
1.2
76
8.9
“The systems were asdescribedin METHODS andcontained1 mgof cell protein, 10mM NanNO, and 50 mM NaN,. Total volume was 1.1-1.3ml and headspacevolume wasabout 8 ml.
kinetically effective in NO uptake but produced
a nitrosating
species (presumably
N,O,)
in
sufficient amounts so as to make it useless for the experiments at hand. CrSO,, because of its well known ability to reduce NO to ammonia,
avoided this problem.
None of the traps tested reacted
with N,O, in so far as we could determine.
This work was supported by grant DCB 88-16273 from the National 904
Science Foundation.
Vol.
175,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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.
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