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Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum
BIOCHIMIE, 1978, 60, 339-341.
Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum. Paul-Antoine LESP[NAT, Richard a n d Yves B E R L I E R .
GEI~STER
D~partement de Bioi'ogie, S e r v i c e de Radioagronomie, Centre d'Etudes Nucl~aires de Cadaraehe, B P n ° 1, 13115 Saint-Paul-lez-Duranee, France.
Introduction.
Material and Methods.
The unidirectional ATP-independent hydrogen a s e f o u n d i n Azotobacter c h r o o c o c c u m [1] a n d i n s o m e R h i z o b i u m b a c t e r o i d s [2j h a s b e e n a s s i g n e d t h r e e p o s s i b l e f u n c t i o n s [3] : b y r e o x i d i z i n g H 2 p r o d u c e d b y n i t r o g e n a s e it -would, first r e c y c l e wasted ATP and reducing power, second prevent t t 2 i n h i b i t i o n of n i t r o g e n a s e a c t i v i t y a n d t h i r d utilize excess oxygen and thus help maintain anae r o b i c c o n d i t i o n s n e a r t h e a c t i v e s i t e s of n i t r o g e n a s e . Yet t h e c h a r a c t e r i s t i c s of t h e r e l a t i o n b e t w e e n h y d r o g e n a s e a n d n i t r o g e n a s e a r e so f a r n o t c l e a r l y e s t a b l i s h e d . T h e u s e of s t a b l e i s o t o p e d e u t e r i u m (D~) h a s t h o u g h p r o v e d v e r y f r u i t f u l f o r d e t e r m i n i n g h y d r o g e n a s e a c t i v i t y [2, 4] a n d at t h e same time revealing the hydrogen evolving capac i t y of n i t r o g e n a s e i n a e r o b i c fixers, w h i c h otherwise can only be demonstrated in vivo t h r o u g h h y d r o g e n a s e i n h i b i t o r s [5].
The mass-spectrometer, a n ¢ Atlas >> CH~ type was equipped w i t h a n a u t o m a t e d system for peak i n t e n s i t y m e a s u r e m e n t s [10]. The ion-source was directly connected t h r o u g h a stainless-steel v a c u u m line to a react i o n vessel (fig. 1) consisting of a plexiglas cylinder 15 m m wide a n d 60 m m h i g h w i t h a t h e r m o s t a t e d jacket. The volume of the vessel is a d j u s t a b l e b e t w e e n 3 a n d 8 m l b y w a y of a p l u n g e r fitted w i t h t h r e e 0-rings. The m e d i u m inside the vessel can be sparged w i t h a g a s - m i x t u r e b y m e a n s of a n h y p o d e r m i c needle i n t r o d u c e d t h r o u g h p u n c t u r e - b o r e s near the b o t t o m end. T h e n the p l u n g e r is lowered to e l i m i n a t e the excess a t m o s p h e r e t h r o u g h the central canal a n d t i g h t e n e d by a screw.
I n s u c h s t u d i e s , a d i f f i c u l t y lies i n t h a t t h e changes in atmosphere composition represent but a d e l a y e d a n d w e a k e n e d p i c t u r e of -what r e a l l y h a p p e n s at t h e e n z y m e sites. A b e t t e r a c c u r a c y c o u l d b e e x c e p t e d if gas m e a s u r e m e n t s w e r e p e r f o r m e d w i t h i n t h e l i q u i d . I n t h a t -way, a n a m p e r o metric method for measuring dissolved H 2 by m e a n s of a C l a r k e l e c t r o d e [6] h a s b e e n s u c c e s s fully applied to follow H 2 metabolism in bacter o i d s [7] a n d i n R h o d o p s e u d o m o n a s capsu(ata [8]. W i t h a v i e w to e x t e n d i n g s u c h d e t e r m i n a t i o n s t o other gas compounds involved in enzyme activit i e s a d i r e c t m a s s - s p e c t r o m e t r i c m e t h o d [9] allowing measurements of g a s e s d i s s o l v e d i n t h e liquid phase has been applied in this laboratory to t h e s t u d y of t h e r e l a t i o n s h i p b e t w e e n r e s p i r a tion, hydrogenase and nitrogenase activities in
Azotobacter c h r o o c o c c u m .
The m e d i u m is c o n t i n u o u s l y stirred t h a n k s to a m a g n e t i c b a r ; a f r a c t i o n of dissolved gases diffuses t h r o u g h a teflon m e m b r a n e 12.5 Itm thick then, a f t e r w a t e r - v a p o r h a s b e e n condensed in a l i q u i d n i t r o g e n trap, is a d m i t t e d into the ion source. In t h e s e condit i o n s t h e m a s s - s p e c t r o m e t e r signals are p r o p o r t i o n a l to the c o n c e n t r a t i o n of gas c o m p o n e n t s in the liquid phase. The m i c r o o r g a n i s m used in these experiments, a n
Azotobacter chroococcum s t r a i n isolated i n Dr. Dommergues' l a b o r a t o r y , was g r o w n on a B6 Burk's m e d i u m [11] s u p p l e m e n t e d w i t h 5 per cent glucose in a c o n t i n u o u s culture f e r m e n t e r . T h o u g h c o n t i n u o u s l y aerated, the culture was O~-limited as indicated b y a n oxygen-electrode. 8 ml of t h i s culture (optical density close to 0.5 at 570 nm) were t r a n s f e r r e d to t h e reaction vessel t h e n sparged w i t h a m i x t u r e of e i t h e r 0.8 Ar a n d 0 . 2 0 ~ or 0.7 Ar, 0.2 O~ a n d 0.1 D,2 u n t i l t h e a t m o s p h e r e - s o l u t i o n e q u i l i b r i u m was reached as i n d i cated b y t h e levelling off of peaks 32 a n d 4. After the excess a t m o s p h e r e h a d been excluded as described above, the changes in peak heights of O~ (32), D2 (4) a n d H2 (2') were followed e i t h e r separately in one single e x p e r i m e n t or separately in repeated experim e n t s on t h e same sample. All p a r t i a l p r e s s u r e s of dissolved gases were expressed i n a t m o s p h e r e units, the t e m p e r a t u r e of the culture being kept at 30°C t h r o u g h o u t t h e experiments. The s e l f - c o n s u m p t i o n of the mass-spectrometer, a first order f u n c t i o n of gas pressure, was d e t e r m i n e d on w a t e r or u n i n o c u l a t e d m e d i u m sparged in the same conditions.
P. A. Lespinat and coll.
340
Results and Discussion. T h e changes in dissolved gases c o n c e n t r a t i o n are s h o w n in figure 2. T h e r e s p i r a t i o n rate w a s significantly h i g h e r in t h e p r e s e n c e of 0.0.1 D 2 t h a n in its absence ( r e s p e c t i v e l y ,0.'04 and 0.03
of the Ar, 02, D 2 a t m o s p h e r e kept above the culture at the end of sparging. W h i l e 0 e c o n s u m p t i o n in the p r e s e n c e of CO + Cull 2 d e c r e a s e d only slightly, the v e l o c i t y constant of the final D 2 uptake w a s l o w e r e d 6 to 7 times. Yet, the same p a t t e r n was o b s e r v e d for H 2 e v o l u t i o n w h i c h took place in e i t h e r case at the same 0 2 pressure. F r o m these results it can be c o n c l u d e d that H a evolution is not related to the cessation of h y d r o genase a c t i v i t y but to the starting of n i t r o g e n a s e a c t i v i t y and that both activities are directly, yet inversely, d e p e n d i n g on the pO e level. T h i s is in
1¢
2.0 ¸
2.0
5 6
tO °~ ,.,-, 0 > (9 ~)
Fro. 1. - - Reaction oessel for continuous measurement of gases dissolved in liquid phase.
1. Tightening serew with an 0-ring. 2. Plunger. 3. Thermoregulated jaeket. 4. 0-rings. 5. Magnetic bar. 6. Proteetive perforated disk. 7. Teflon membrane. 8. Fritted steel holder. 9. Outlet towards mass-spectrometer. 10. Inlet for sparging gas or introdueing reagents.
a t m o s p h e r e 02 c o n s u m e d p e r minute). Meanwhile, D 2 c o n s u m p t i o n e x h i b i t e d a t y p i c a l dual k i n e t i c s p a t t e r n i n v o l v i n g a faster and a s l o w e r c o m p o n e n t . P l o t t i n g the data along a s e m i l o g a r i t h m i c coordinate system (insert of fig. 2), a straight line was o b t a i n e d for the s l o w e r c o m p o n e n t , p a r a l l e l i n g m a s s - s p e c t r o m e t e r self-consumption. T h e actual D u uptake t h r o u g h the u n i d i r e c t i o n a l h y d r o g e n a s e was thus r e p r e s e n t e d by the faster c o m p o n e n t and ceased almost c o m p l e t e l y after five m i n u t e s w h i l e oxygen p r e s s u r e h a d d e c l i n e d to about 0,.01 atmosphere. At the v e r y same time H 2 e v o l u t i o n o c c u r e d abruptly, but only w h e n D 2 was present. In a n o t h e r set of e x p e r i m e n t s , the different kinetics of gas p r o d u c t i o n and uptake have been comp a r e d in the p r e s e n c e or the absence of a h y d r o genase i n h i b i t i n g m i x t u r e c o m p o s e d of 0.5 ml CO plus ,0.5 ml Curio i n t r o d u c e d into about 3 ml BIOCHIMIE, 1978, 60, n ° 3.
1.0 1.5
"5"
.3
1.0
G) .a¢ t~
5
10
15
t). (/) (~ 01
.5
\ \
5
10
time
(minutes)
15
FIG. 2. Time course of 02 and D e consumption and H~ evolution after spargzng the culture w i t h 0.7 At, 0 . 2 0 ~ and 0.1 D=. • H2 evolution. [] 02 uptake in the -
-
presence of D=. O 02 uptake in the absence of D2. • D2 uptake. Insert : semilogarithmie plot of D~ consumption. D~ uptake by hydrogenase is the difference between the first part of the curve (full line) and the slower component extrapolated towards time zero (dotted line). The ordinate scale units are respectively 0.1 atmosphere O~, 0.06 atmosphere D2 and 0.005 atmosphere H~.
a c c o r d a n c e w i t h the (( s w i t c h off-switch on)> m o d e l put f o r w a r d by Droszd and Postgate [12] to e x p l a i n the ¢ conformational>> p r o t e c t i o n of n i t r o g e n a s e against excess 02 : in A z o t o b a c t e r c h r o o c o c c u m p r e v i o u s l y g r o w n at 0.0'9 a t m o s p h e r e
Respiration
and hydrogenase-nitrogenase
02,
n i t r o g e n a s e s w i t c h e d off w h e n pO 2 w a s r i s e n f r o m 0:05 to 0.2 a t m o s p h e r e , t h e n s w i t c h e d o n again upon restoring the initial conditions. Here, however, the model applies to both hydrogenase and nitrogenase activities, the former being s w i t c h e d off a n d t h e l a t t e r s w i t c h e d o n w h e n p O 2 is l o w e r e d t h r o u g h r e s p i r a t i o n . T h i s is c o n s i s t e n t with a regulation process by hydrogenase of n i t r o g e n a s e a c t i v i t y a c c o r d i n g to t h e o x y g e n s t a t u s of t h e c u l t u r e . B o t h e n z y m e s w o u l d t h e r e f o r e h a v e v e r y c l o s e l o c a t i o n s i n t h e b a c t e r i a l cell. Among the different functions proposed by D i x o n [3] f o r t h e u n i d i r e c t i o n a l h y d r o g e n a s e of a e r o b i c f i x e r A z o t o b a c t e r c h r o o c o c c u m , its p r o t e c t i v e r o l e b y s c a v e n g i n g o x y g e n at t h e v i c i n i t y of t h e a c t i v e s i t e s of n i t r o g e n a s e is t h u s e m p h a sized.
Acknowledgements. W e are indebted to Dr Dommergaes for k i n d l y supplying the bacterial strain.
BIOCHIMIE, 1978, 60, n ° 3.
relationship.
341
This work was partly supported by f u n d s f r o m the <~DM~gation 6 la Recherche S c i e n t i f i q u e et Technique >) (A.C.C. <~F i x a t i o n de l'Azole ~).
REFERENCES. 1. H y n d m a n , L. A., Burris, R. H. a Wilson, P. W. (1953) J. Bact., 65, 522-531. 2. Dixon, R. O. D. (1968) Arch. Mikrobiol., 62, 272-283. 3. Dixon, R. O. R. (1972) Arch. Mikrobiol., 85, 193-201. 4. Berlier, Y. M. & Lespinat, P. A. (1978) FEMS Microbiol. Letters, in press. 5. Smith, L. A., Hill, S. & Yates, M. G. (1976) Nature, 262, 210-211. 6. Wang, R., Healey, F. P. & Myers, J. (1971) P l a n t Physiol.. 48, 108-110. 7. Schubert, K. R. & Evans, H. J. (1976) Proc. Nat. Acad. Sci., USA, 73, 1207-1211. 8. Kelley, B. C., Meyer, C. M., Gandy, C. & Vignais, P. M. (1977) FEBS Letters, 81, 281-285. 9. Hoch, G. a Kok, B. (1963) Arch. Bioehem. Biophys., 101, 160-170. 10. Gerster, R. (1971) Echanges isotopiques e n t r e CO: et H~O et cin6tiques de m a r q u a g e de l'oxyg6ne p h o t o s y n t h 6 t i q u e . Th6se de Doctoral d'Etat. Universit6 d'Aix-Marseille, Centre de L u m i n y . 11. Dalton, H. a Postgate, J. R. (1969) J. Gen. Microbiol., 54, 463-473. 12. Droszd., J. a Postgate, J. R. (1970) J. Gen. Microbiol., 60, 427-429.
Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum. Paul-Antoine LESP[NAT, Richard a n d Yves B E R L I E R .
GEI~STER
D~partement de Bioi'ogie, S e r v i c e de Radioagronomie, Centre d'Etudes Nucl~aires de Cadaraehe, B P n ° 1, 13115 Saint-Paul-lez-Duranee, France.
Introduction.
Material and Methods.
The unidirectional ATP-independent hydrogen a s e f o u n d i n Azotobacter c h r o o c o c c u m [1] a n d i n s o m e R h i z o b i u m b a c t e r o i d s [2j h a s b e e n a s s i g n e d t h r e e p o s s i b l e f u n c t i o n s [3] : b y r e o x i d i z i n g H 2 p r o d u c e d b y n i t r o g e n a s e it -would, first r e c y c l e wasted ATP and reducing power, second prevent t t 2 i n h i b i t i o n of n i t r o g e n a s e a c t i v i t y a n d t h i r d utilize excess oxygen and thus help maintain anae r o b i c c o n d i t i o n s n e a r t h e a c t i v e s i t e s of n i t r o g e n a s e . Yet t h e c h a r a c t e r i s t i c s of t h e r e l a t i o n b e t w e e n h y d r o g e n a s e a n d n i t r o g e n a s e a r e so f a r n o t c l e a r l y e s t a b l i s h e d . T h e u s e of s t a b l e i s o t o p e d e u t e r i u m (D~) h a s t h o u g h p r o v e d v e r y f r u i t f u l f o r d e t e r m i n i n g h y d r o g e n a s e a c t i v i t y [2, 4] a n d at t h e same time revealing the hydrogen evolving capac i t y of n i t r o g e n a s e i n a e r o b i c fixers, w h i c h otherwise can only be demonstrated in vivo t h r o u g h h y d r o g e n a s e i n h i b i t o r s [5].
The mass-spectrometer, a n ¢ Atlas >> CH~ type was equipped w i t h a n a u t o m a t e d system for peak i n t e n s i t y m e a s u r e m e n t s [10]. The ion-source was directly connected t h r o u g h a stainless-steel v a c u u m line to a react i o n vessel (fig. 1) consisting of a plexiglas cylinder 15 m m wide a n d 60 m m h i g h w i t h a t h e r m o s t a t e d jacket. The volume of the vessel is a d j u s t a b l e b e t w e e n 3 a n d 8 m l b y w a y of a p l u n g e r fitted w i t h t h r e e 0-rings. The m e d i u m inside the vessel can be sparged w i t h a g a s - m i x t u r e b y m e a n s of a n h y p o d e r m i c needle i n t r o d u c e d t h r o u g h p u n c t u r e - b o r e s near the b o t t o m end. T h e n the p l u n g e r is lowered to e l i m i n a t e the excess a t m o s p h e r e t h r o u g h the central canal a n d t i g h t e n e d by a screw.
I n s u c h s t u d i e s , a d i f f i c u l t y lies i n t h a t t h e changes in atmosphere composition represent but a d e l a y e d a n d w e a k e n e d p i c t u r e of -what r e a l l y h a p p e n s at t h e e n z y m e sites. A b e t t e r a c c u r a c y c o u l d b e e x c e p t e d if gas m e a s u r e m e n t s w e r e p e r f o r m e d w i t h i n t h e l i q u i d . I n t h a t -way, a n a m p e r o metric method for measuring dissolved H 2 by m e a n s of a C l a r k e l e c t r o d e [6] h a s b e e n s u c c e s s fully applied to follow H 2 metabolism in bacter o i d s [7] a n d i n R h o d o p s e u d o m o n a s capsu(ata [8]. W i t h a v i e w to e x t e n d i n g s u c h d e t e r m i n a t i o n s t o other gas compounds involved in enzyme activit i e s a d i r e c t m a s s - s p e c t r o m e t r i c m e t h o d [9] allowing measurements of g a s e s d i s s o l v e d i n t h e liquid phase has been applied in this laboratory to t h e s t u d y of t h e r e l a t i o n s h i p b e t w e e n r e s p i r a tion, hydrogenase and nitrogenase activities in
Azotobacter c h r o o c o c c u m .
The m e d i u m is c o n t i n u o u s l y stirred t h a n k s to a m a g n e t i c b a r ; a f r a c t i o n of dissolved gases diffuses t h r o u g h a teflon m e m b r a n e 12.5 Itm thick then, a f t e r w a t e r - v a p o r h a s b e e n condensed in a l i q u i d n i t r o g e n trap, is a d m i t t e d into the ion source. In t h e s e condit i o n s t h e m a s s - s p e c t r o m e t e r signals are p r o p o r t i o n a l to the c o n c e n t r a t i o n of gas c o m p o n e n t s in the liquid phase. The m i c r o o r g a n i s m used in these experiments, a n
Azotobacter chroococcum s t r a i n isolated i n Dr. Dommergues' l a b o r a t o r y , was g r o w n on a B6 Burk's m e d i u m [11] s u p p l e m e n t e d w i t h 5 per cent glucose in a c o n t i n u o u s culture f e r m e n t e r . T h o u g h c o n t i n u o u s l y aerated, the culture was O~-limited as indicated b y a n oxygen-electrode. 8 ml of t h i s culture (optical density close to 0.5 at 570 nm) were t r a n s f e r r e d to t h e reaction vessel t h e n sparged w i t h a m i x t u r e of e i t h e r 0.8 Ar a n d 0 . 2 0 ~ or 0.7 Ar, 0.2 O~ a n d 0.1 D,2 u n t i l t h e a t m o s p h e r e - s o l u t i o n e q u i l i b r i u m was reached as i n d i cated b y t h e levelling off of peaks 32 a n d 4. After the excess a t m o s p h e r e h a d been excluded as described above, the changes in peak heights of O~ (32), D2 (4) a n d H2 (2') were followed e i t h e r separately in one single e x p e r i m e n t or separately in repeated experim e n t s on t h e same sample. All p a r t i a l p r e s s u r e s of dissolved gases were expressed i n a t m o s p h e r e units, the t e m p e r a t u r e of the culture being kept at 30°C t h r o u g h o u t t h e experiments. The s e l f - c o n s u m p t i o n of the mass-spectrometer, a first order f u n c t i o n of gas pressure, was d e t e r m i n e d on w a t e r or u n i n o c u l a t e d m e d i u m sparged in the same conditions.
P. A. Lespinat and coll.
340
Results and Discussion. T h e changes in dissolved gases c o n c e n t r a t i o n are s h o w n in figure 2. T h e r e s p i r a t i o n rate w a s significantly h i g h e r in t h e p r e s e n c e of 0.0.1 D 2 t h a n in its absence ( r e s p e c t i v e l y ,0.'04 and 0.03
of the Ar, 02, D 2 a t m o s p h e r e kept above the culture at the end of sparging. W h i l e 0 e c o n s u m p t i o n in the p r e s e n c e of CO + Cull 2 d e c r e a s e d only slightly, the v e l o c i t y constant of the final D 2 uptake w a s l o w e r e d 6 to 7 times. Yet, the same p a t t e r n was o b s e r v e d for H 2 e v o l u t i o n w h i c h took place in e i t h e r case at the same 0 2 pressure. F r o m these results it can be c o n c l u d e d that H a evolution is not related to the cessation of h y d r o genase a c t i v i t y but to the starting of n i t r o g e n a s e a c t i v i t y and that both activities are directly, yet inversely, d e p e n d i n g on the pO e level. T h i s is in
1¢
2.0 ¸
2.0
5 6
tO °~ ,.,-, 0 > (9 ~)
Fro. 1. - - Reaction oessel for continuous measurement of gases dissolved in liquid phase.
1. Tightening serew with an 0-ring. 2. Plunger. 3. Thermoregulated jaeket. 4. 0-rings. 5. Magnetic bar. 6. Proteetive perforated disk. 7. Teflon membrane. 8. Fritted steel holder. 9. Outlet towards mass-spectrometer. 10. Inlet for sparging gas or introdueing reagents.
a t m o s p h e r e 02 c o n s u m e d p e r minute). Meanwhile, D 2 c o n s u m p t i o n e x h i b i t e d a t y p i c a l dual k i n e t i c s p a t t e r n i n v o l v i n g a faster and a s l o w e r c o m p o n e n t . P l o t t i n g the data along a s e m i l o g a r i t h m i c coordinate system (insert of fig. 2), a straight line was o b t a i n e d for the s l o w e r c o m p o n e n t , p a r a l l e l i n g m a s s - s p e c t r o m e t e r self-consumption. T h e actual D u uptake t h r o u g h the u n i d i r e c t i o n a l h y d r o g e n a s e was thus r e p r e s e n t e d by the faster c o m p o n e n t and ceased almost c o m p l e t e l y after five m i n u t e s w h i l e oxygen p r e s s u r e h a d d e c l i n e d to about 0,.01 atmosphere. At the v e r y same time H 2 e v o l u t i o n o c c u r e d abruptly, but only w h e n D 2 was present. In a n o t h e r set of e x p e r i m e n t s , the different kinetics of gas p r o d u c t i o n and uptake have been comp a r e d in the p r e s e n c e or the absence of a h y d r o genase i n h i b i t i n g m i x t u r e c o m p o s e d of 0.5 ml CO plus ,0.5 ml Curio i n t r o d u c e d into about 3 ml BIOCHIMIE, 1978, 60, n ° 3.
1.0 1.5
"5"
.3
1.0
G) .a¢ t~
5
10
15
t). (/) (~ 01
.5
\ \
5
10
time
(minutes)
15
FIG. 2. Time course of 02 and D e consumption and H~ evolution after spargzng the culture w i t h 0.7 At, 0 . 2 0 ~ and 0.1 D=. • H2 evolution. [] 02 uptake in the -
-
presence of D=. O 02 uptake in the absence of D2. • D2 uptake. Insert : semilogarithmie plot of D~ consumption. D~ uptake by hydrogenase is the difference between the first part of the curve (full line) and the slower component extrapolated towards time zero (dotted line). The ordinate scale units are respectively 0.1 atmosphere O~, 0.06 atmosphere D2 and 0.005 atmosphere H~.
a c c o r d a n c e w i t h the (( s w i t c h off-switch on)> m o d e l put f o r w a r d by Droszd and Postgate [12] to e x p l a i n the ¢ conformational>> p r o t e c t i o n of n i t r o g e n a s e against excess 02 : in A z o t o b a c t e r c h r o o c o c c u m p r e v i o u s l y g r o w n at 0.0'9 a t m o s p h e r e
Respiration
and hydrogenase-nitrogenase
02,
n i t r o g e n a s e s w i t c h e d off w h e n pO 2 w a s r i s e n f r o m 0:05 to 0.2 a t m o s p h e r e , t h e n s w i t c h e d o n again upon restoring the initial conditions. Here, however, the model applies to both hydrogenase and nitrogenase activities, the former being s w i t c h e d off a n d t h e l a t t e r s w i t c h e d o n w h e n p O 2 is l o w e r e d t h r o u g h r e s p i r a t i o n . T h i s is c o n s i s t e n t with a regulation process by hydrogenase of n i t r o g e n a s e a c t i v i t y a c c o r d i n g to t h e o x y g e n s t a t u s of t h e c u l t u r e . B o t h e n z y m e s w o u l d t h e r e f o r e h a v e v e r y c l o s e l o c a t i o n s i n t h e b a c t e r i a l cell. Among the different functions proposed by D i x o n [3] f o r t h e u n i d i r e c t i o n a l h y d r o g e n a s e of a e r o b i c f i x e r A z o t o b a c t e r c h r o o c o c c u m , its p r o t e c t i v e r o l e b y s c a v e n g i n g o x y g e n at t h e v i c i n i t y of t h e a c t i v e s i t e s of n i t r o g e n a s e is t h u s e m p h a sized.
Acknowledgements. W e are indebted to Dr Dommergaes for k i n d l y supplying the bacterial strain.
BIOCHIMIE, 1978, 60, n ° 3.
relationship.
341
This work was partly supported by f u n d s f r o m the <~DM~gation 6 la Recherche S c i e n t i f i q u e et Technique >) (A.C.C. <~F i x a t i o n de l'Azole ~).
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