The in vitro reduction of N_2 is a complex process involving at least six different reactants: two proteins [1,2] for which the names azoferredoxin (AzoFd) and molybdoferredoxin (MoFd) have been proposed[3], an electron source, the electron acceptor, ATP[4], and Mg2+[5-7]. One of the goals of research in this area is to define the orderly and quantitative participation of these reactants leading to the reduction of the electron acceptor with concomitant breakdown of ATP to ADP and inorganic phosphate[7]. 



The work described in this paper shows that (1) AzoFd reversibly binds both ATP, a reactant in N2 reduction, and ADP, a specific inhibitor of N2 reduction, and (2) MoFd reversibly binds cyanide, which is also reduced by the N_2-reducing system. It is suggested that the binding of ATP and of cyanide are partial reactions of the N_2-reducing system

Bui, P. T.

Mortenson, L. E.

English

Caltech Authors - Main

MECHANISM OF THE ENZYMIC REDUCTION OF N2: THE
BINDING OF ADENOSINE 5'-TRIPHOSPHATE AND CYANIDE TO
THE NrREDUCING SYSTEM*
BY P. T. BuIt AND L. E. MORTENSON
DEPARTMENT OF BIOLOGICAL SCIENCES, PURDUE UNIVERSITY
Communicated by Martin D. Kamen, August 27, 1968
The in vitro reduction of N2 is a complex process involving at least six different
reactants: two proteins" 2 for which the names azoferredoxin (AzoFd) and
molybdoferredoxin (MoFd) have been proposed,3 an electron source, the electron
acceptor, ATP,4 and Mg2+. 5-7 One of the goals of research in this area is to
define the orderly and quantitative participation of these reactants leading to
the reduction of the electron acceptor with concomitant breakdown of ATP to
ADP and inorganic phosphate.'
The work described in this paper shows that (1) AzoFd reversibly binds both
ATP, a reactant in N2 reduction, and ADP, a specific inhibitor of N2 reduction,
and (2) MoFd reversibly binds cyanide, which is also reduced by the N2-reducing
system. It is suggested that the binding of ATP and of cyanide are partial
reactions of the N2-reducing system.
Methods.-Buffers: 2-Amino-2-(hydroxymethyl)-1 ,3-propanediol, dissolved in 0.5
equivalents of HCl, was used in cyanide-binding experiments at 50 mM (pH 8.1). Sodium
piperazine-N,N'-bis (2-ethanesulfonic acid) (Calbiochem), dissolved in 0.4 equivalents of
LiOH, was used in all other experiments at 50 mMI (pH 6.6).
Biochemicals: Sodium salts of ATP, AMP, CDP, GDP, IDP, and UDP were purchased
from Sigma. Trilithium ADP was purchased from Calbiochem. Uniformly labeled "4C-
ATP and 14C-ADP were purchased from Schwarz BioResearch. K'4CN was purchased
from International Chemical and Nuclear Corporation.
Proteins: AzoFd (approximate purity 90%), prepared by a modifications8 of the method
of Mortenson et al.,3 was a gift from E. Moustafa. The molecular weight of 4 X 104 used
in all binding calculations was estimated by Sephadex chromatography. MoFd, purified
to a constant Mo/Fe/protein by a modification' of the method of Mortenson et al.,3 was a
gift from J. A. Morris. The molecular weight of 1.6 X 105 used in binding calculations
was estimated by Sephadex chromatography.
Cyanide reduction: Cyanide is an excellent substrate for studies of the Nrreducing
system for several reasons: (1) it can be handled easily because it is a solid, (2) it is re-
duced by the N2-reducing system to ammonia and methane,'0 which can be quantitated
rapidly by gas chromatography, and (3) it can be readily obtained in a radioactive form,
K'4CN. Cyanide solutions were prepared anaerobically and stored under hydrogen at
-20°. The reduction was carried out at 22° in glass vials (8 ml vol) sealed with rubber
serum stoppers and equipped with magnetic stirring bars. The gas phase in the vials
was evacuated and replaced by hydrogen. AzoFd, MoFd, and the electron donor dithio-
nitell were added anaerobically since they are extremely sensitive to oxygen. The reaction
was started by the addition of ATP, and stopped when necessary by the addition of 0.05
ml of 10 N KOH. Unless indicated otherwise, the vials contained in 2.0 ml total volume
the following buffered reaction mixture: 0.2 mg AzoFd, 0.8 mg MoFd, 4.0 mM KCN,
4.0 mM dithionite (Na,2S,04), 2.0 mM ATP, and 2.0 mM MgC12. Gas samples were re-
moved at appropriate time intervals and analyzed for methane by gas chromatography
using a Porapak R column,12 1.8 mm diameter X 40 cm, at 220.
Detection of binding by gel Jiltration: An anaerobic column of 2.4 cm diameter was
packed with 6.0 gm of Sephadex G-50, then washed with at least one bed volume of de-
aerated equilibrating buffer prior to the application and elution of the protein sample at
1021
BIOCHEMISTRY: BUI AND MORTENSON
constant rate (60 ml/hr). K1"CN, 14C-ATP, or 14C-ADP were added to the equilibrating
buffer at the low concentration of 0.1 mM since a high signal (bound substrate or inhibitor)
to noise (background of free substrate or inhibitor) ratio was necessary for a quantitative
estimation of substrate or inhibitor binding by the test proteins. MgCl2 (0.5 mM) and
dithionite (4.0 mM) were also present in the experiments testing ATP and AMP for bind-
ing to AzoFd in competition with ADP. The effluent was collected in 1.0-ml samples
and analyzed for protein by the biuret method, and for radioactivity, which was measured
at 220 in a liquid scintillation counter (Beckman model LS-150) by mixing 0.5 ml of the
column effluent with 7.0 ml of a scintillation fluid containing 5.0 gm of 2,5-diphenyloxazole
and 50 gm of naphthalene per liter of 1,4-dioxane.
Results.-ADP inhibition of cyanide reduction: ADP has been shown to
inhibit reductant-dependent ATP hydrolysis,5 acetylene reduction,6 ATP-de-
pendent H2 evolution,7 and N2 reduction,7 all of which are catalytic activities of
the N2-reducing system. The inhibition was not a result of ADP decreasing the
amount of Mg2+ available,6' 7 for example by forming a (Mg-ADP) complex.
Increasing concentrations of ADP affect the rate of cyanide reduction (Fig. 1 or
ATP-dependent H2 evolution7 in a similar manner. When the data from the
experiment on the inhibition of cyanide reduction by ADP (Fig. 1) were plotted
as log (v/(vo- v)) vs. log ADP, 3 the slope of the resulting curve (Fig. 2) indicated
that the inhibition required the participation of three ADP molecules per cyanide-
reducing unit. The specific inhibition of the N2-reducing activity in vitro by an
end product in ammonia synthesis, ADP (Table 1), suggests that the inhibition
operates in vivo as an energy-conserving device. When the supply of energy in
the cell becomes limiting, the resulting ADP build-up will decelerate the break-
down of ATP by the N2-reducing system.
Binding of cyanide to MoFd: Earlier attempts to define the role of MIoFd and
AzoFd in N2 reduction'4-'6 were unsuccessful5 7, 17 because the techniques used5
100
0
0
2 3 4 -3.0 -2.5
ADP (mM) log ADP
FIG. 1.-ADP inhibition of cyanide FIG. 2.-ADP inhibition of cyanide reduction.
reduction. Cyanide reduction was assayed Initial rates of cyanide reduction in the presence
as described in Methods, in the presence (v) or absence (vo) of ADP were taken from
of increasing concentrations of ADP. One Fig. 1. The concentration of ADP is in moles/
hundred per cent activity corresponds to liter. The slope of the curve, n = 2.8, corre-
an initial rate of 60 mprmoles methane sponds approximately to the number of ADP
produced/min. molecules required to inactivate the cyanide-
reducing unit. The number of ATP molecules
required to activate the acetylene-reducing unit
is 2 or 3, depending on whether ADP is absent
or present.6
1022 PROC. N. A. S.
BIOCHEMISTRY: BUI AND MORTENSON
TABLE 1. The specificity of the inhibition of cyanide reduction by ADP.
Additions Activity (%)
None
ADP (4.0 mM)
CDP (4.7 mM)
GDP (4.1 mM)
IDP (4.3 mM)
UDP (5.2 mM)
AMP (4.0 mM) + Na4P207 (4.0 mM)
K2HP04 (4.0 mM) + Na2HAsO4 (4.0 mM)
100
6
91
86
94
95
92
94
Cyanide reduction was assayed (as described in Methods) in the presence of various nucleoside
phosphates at the concentrations shown. One hundred per cent activity corresponded to an initial
rate of 60 mpmoles methane produced/min.
were incapable of detecting reversible associations between proteins and sub-
strates. This led us to adapt for use under anaerobic conditions a simple method
capable of detecting reversible as well as irreversible associations between two
chemical species of different molecular weights.18 The method consists of first
equilibrating a Sephadex G-50 column with buffer containing a substrate of the
Nrreducing system (cyanide or ATP), or an inhibitor (ADP), and any other
necessary reactant (e.g. MgCl2). Next the test protein is passed through the
column. If the protein has an affinity for the substrate (or inhibitor), one should
be able to detect an accumulation of substrate (or inhibitor) above the equilib-
rium level and coincident with the protein peak. In the absence of any inter-
action, the equilibrium concentration of the substrate (or inhibitor), constant
throughout the column, should not be disturbed by passage of the protein band.
When MoFd and AzoFd were tested in this manner, the elution pattern of pro-
tein and radioactivity showed binding of '4CN- to MoFd (Fig. 3), but not to
AzoFd (Fig. 4). The binding was not enhanced by the addition of other reac-
20
effluent (ml)
140 -
0
0)
10
120 E
E
z
0
100
0
I
E
0
0-cL
0
15 20
effluent (ml)
140
(0
0,
0
120 E
E
Iz
100
FIG. 3.-Binding of cyanide by MoFd.
The equilibrating buffer for the Sephadex
column (see Methods) contained 0.1 mM
U4CN- (at this concentration of cyanide,
the ratio of cyanide bound per MoFd was
0.6).
FIG. 4.-Lack of binding of cyanide by
AzoFd. The equilibrating buffer for the
Sephaddx column (see Methods) contained
0.1 mM 14CN-. Identical results were
obtained when 4.0 mM dithionite, 2.0
mM ATP, and 2.0 mM MgCl2 were also
added to the buffer.
0
-9
coE
0-
0
Io
)_2#°° °0R~~~~~~-I
0
0
000
-
-1
I I
VOL. 61, 1968 1023
BIOCHEMISTRY: BUI AND MORTENSON
tants in N2 reduction, singly or in any combination (the binding of cyanide by
MIoFd in the presence of AzoFd, ATP, Ig2+, and dithionite was not tested since
cyanide is converted to methane and ammonia under these conditions). The
binding was reversible since no radioactivity could be detected with I\IoFd which
had been incubated with '4CN- and then passed through Sephadex G-50 to
separate unbound 14CN-. Efforts to detect N2 binding by MoFd, using anaero-
bic spectrophotometric techniques, are in progress, and will be reported else-
where.9
Binding of ATP and ADP to AzoFd: Since ATP and ADP are chemically
similar, it is reasonable to expect that they might bind to the same site on the N2-
reducing system. This site must be on AzoFd since AzoFd binds both ATP
(Fig. 5) and ADP (Fig. 6), whereas MoFd binds neither. Like the binding of
-16014
0 3
'4-APanl.m gl20obidn1CA
E~~~~~~~~~~~~~7
0~~~~~~~~~~~~~~~~~~~
0
Mg~~~~~~~~l2.~~ ~ ~
A.P th idn fAPi.o)euraddM2.Clultoswt h
C-)~~~~~~~~~~~~~~E
0 1~~~~~~~~~~
0 L.. -100100
15 20 15 20
effluent (ml) effluent (Ml
FIG. 5.-Binding of ATP to AzoFd. FIG. 6.-Binding of ADP to AzoFd.
The equilibrating buffer for the Sephadex The equilibrating buffer f r the Sephadex
column (see Methods) contained 0.1 mM column (see Methodp)contained 0.1 mM
14C-ATP, and 0.5 mM Mg9l2. No binding 14C-ADP.
could be detected in the absence of added
MgCl2.
ATP, the binding of ADP by AzoFd was reversible, but unlike the binding of
ATP, the binding of ADP did not require added Mg2t. Calculations with the
data available indicate that under the experimental conditions described, AzoFd
binds both ATP and ADP to the same extent (0.4 ATP or ADP per AzoFd).
The low ratio of ATP (or ADP) bound per AzoFd is a consequence of the neces-
sity (see Methods) of using a low ATP concentration (5% of the optimal concen-
tration for N2 reduction') in binding experiments. In addition, if both ATP and
ADP bind at the same site, they should compete in binding to AzoFd. In fact,
the amount of '4C-ADP bound per AzoFd decreased to less than 15 per cent when
the equilibrating buffer contained either 1.0 mM 12C-ATP or 1.0 mM 12C-ADP in
addition to the 0.1 mM 14C-ADP. In contrast, 1.0 mM AMP had no effect on
the binding of 0.1 mM ADP, which suggests that AzoFd either does not bind
AMP or does not bind AMP at the ADP (or ATP) site.
Binding of ADP to the N2-reducing system: The binding of ATP by the N2-
1024 PROC. N. A. S.
BIOCHEMISTRY: BUI AND MORTENSON
reducing system was not examined since any interpretation would be complicated
by a simultaneous conversion of ATP to ADP and Pi. However, the binding of
ADP by the N2-reducing system can be showii (Fig. 7) since ADP is not metabo-
lized by the system. When the binding of ADP by AzoFd was compared to the
binding by the combination of AzoFd and MoFd (the N2-reducing system), it was
clear that MIoFd did not affect the quantity of ADP bound per AzoFd although
it retarded the dissociation of the (ADP AzoFd) complex. This sluggish dis-
sociation accounts for the ADP curve's being skewed toward the back of the pro-
tein curve (Fig. 7).
Discussion.-Significant binding of ADP by the N2-reducing system is pre-
dicted on the basis that (1) the system is responsible for at least 80 per cent of the
ATPbreakdown catalyzed byN2-reducing clostridial extracts;' (2) thisATP break-
down is inhibited by ADP;5 and (3) the system has the same number of binding
sites (Fig. 2) and very nearly the same affinity for ATP and ADP (Figs. 5 and 6).
It is therefore unlikely that a protein contaminant not involved in N2 reduction
could account for all of the ADP binding reported above since AzoFd bound the
same amount of ADP, whether tested alone or as part of the N2-reducing system.
Furthermore, the fact that 1\JoFd did not bind ADP and yet greatly affected the
reversal of ADP binding by the AzoFd fraction (Fig. 7) suggests that a protein
capable of interacting with MoFd, e.g. AzoFd, was responsible for the binding
observed.
In summary, the use of available information on the binding reactions de-
scribed above permits the construction of a more detailed picture of the paths
of all but one of the reactants in the N2-reducing system:
Mg ATP N2 MOFd
I (MgATP.' (Mo Fd N2)
,) Azo Fd)
Azo Fd NH3
AzoFd reductant Pi
ADP
As shown in the diagram, AzoFd binds MIgATP reversibly to form the potentially
reactive complex (MgATP AzoFd). The bond(s) between substrate and pro-
tein in this complex is presently under investigation. The Mg2+ requirement
for N2 reduction is explained by the inability of AzoFd to bind ATP in the ab-
sence of added Mg2+. MoFd is responsible for the reversible binding of the
electron acceptor (N2 or CN-) to form the complex (1\IoFd.N2). Again, the
nature of the bond(s) between substrate and protein is not known. The neces-
sity of having two specific sites, one for the binding of MgATP and the other
for the binding of the electron acceptor (N2 or CN-), cannot be the whole ex-
planation for the requirement for two proteins in N2 reduction since MoFd does
not bind ATP and yet is required for the conversion of AzoFd-bound ATP to
VOL. 61, 1968 1025
BIOCHEMISTRY: BUI AND MORTENSON
-140GK 0 EFIG. 7.-Binding of ADP to AzoFd
_ J \ t s plus MoFd. The equilibrating buffer foi
r t \ _ 120 the Sephadex column (see Methods) con-
E tained 0.1 mM C40-ADP, 0.5 mM MgCl2,
c and 4.0 mM dithionite. AzoFd and MoFd
.'E A/ \ E were combined in a 1.0:1.3 weight ratio.
@ r/ \ a Identical results were obtained when
°OIA1oFd and MoFd were combined in a
*10 1.0:4.0 weight ratio.
15 20
effluent (ml)
ADP and inorganic phosphate. The mechanism by which electrons are accepted
by the Nr-reducing system and used to reduce N2 is unknown, but the fact that no
requirement for dithionite could be demonstrated for the binding of either Mg-
ATP or cyanide suggests that the point of entry for the electrons may be at the
level of (1) (MgATP -AzoFd), (2) (MoFd- N2), or (3) the combination of these
two complexes, rather than at the level of AzoFd or MoFd. One of the products
of ATP metabolism by the N2-reducing system, ADP, is capable of preventing
N2 reduction by acting as an inert ATP analog, i.e. reversibly forming the unreac-
tive complex (ADP AzoFd), thus effectively removing AzoFd which otherwise
would react with MgATP. The lack of inhibition of cyanide reduction by several
other nucleoside phosphates is probably a consequence of their inability to bind
to the ATP site. This is supported by the fact that AMP, which was incapable
of competing with ADP for the ATP binding site, is also inactive in inhibiting
acetylene reduction,6 ATP-dependent H2 evolution,7 reductant-dependent ATP
hydrolysis,7 and cyanide reduction (Table 1).
We appreciate the capable preparative skills of Mrs. Dorothy Sheets and Mrs. Claudette
Polis. We thank Professor W. J. Ray, Jr., for generous advice.
Abbreviations: ATP, adenosine 5'-triphobphate; AMP, adenosine 5'-phosphate; ADP,
CDP, GDP, IDP, and UDP, 5'-diphosphates of adenosine, cytidine, guanosine, inosine, and
uridine; Pi, inorganic orthophosphate.
* Supported by NIH grant AI04865-06.
t Present address: Division of Biology, California Institute of Technology, Pasadena,
California 91109.
1 Mortenson, L. E., in Non-heme Iron Proteins: Role in Energy Conversion, ed. A. San
Pietro (Yellow Springs, Ohio: Antioch, 1965), p. 243.
2 Bulen, W. A., and J. R. LeComte, these PROCEEDINGS, 56, 979 (1966).
3 Mortenson, L. E., J. A. Morris, and D. Y. Jeng, Biochim. Biophys. Acta, 141, 516 (1967).
4Mortenson, L. E., these PROCEEDINGS, 52, 272 (1964).
5 Bui, P. T., Ph.D. Thesis (W. Lafayette, Indiana: Purdue University, 1968).
6 Moustafa, E., and L. E. Mortenson, Nature, 216, 1241 (1967).
7Kennedy, I. R., J. A. Morris, and L. E. Mortenson, Biochim. Biophys. Acta, 153, 777
(1968).
8 Moustafa, E., and L. E. Mortenson, Biochim. Biophys. Acta, in press.
Morris, J. A., and L. E. Mortenson, manuscript in preparation.
'0Hardy, R. W. F., and E. Knight, Jr., Biochim. Biophys. Ada, 139, 69 (1967).
a1
1026 PROC. N. A. S.
VOL. 61, 1968 BIOCHEMISTRY: BUI AND MORTENSON 1027
l Bulen, W. A., R. C. Burns, and J. R. LeComte, these PROCEEDINGS, 53, 532 (1965).
12 Kelly, M., J. R. Postgate, and R. L. Richards, Biochem. J., 102, lc (1967).13 Monod, J., J. P. Changeux, and F. Jacob, J. Mol. Biol., 6, 306 (1963).
14 Bui, P. T., and L. E. Mortenson, Federation Proc., 26, 725 (1967).
15 Kennedy, I. R., and L. E. Mortenson, Bacteriol. Proc., (1967), p. 113.
16 Mortenson, L. E., I. R. Kennedy, and P. T. Bui, Abstracts, Seventh International Congress
Biochemistry (1967), p. 889.
17 Mortenson, L. E., J. A. Morris, and I. R. Kennedy, Bacteriol. Proc. (1968), p. 133.
18 Hummel, J. P., and W. J. Dreyer, Biochim. Biophys. Acta, 65, 530 (1962).


Mechanism of the Enzymic Reduction of N_2: The Binding of Adenosine 5'-Triphosphate and Cyanide to the N_2-reducing System

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Mechanism of the Enzymic Reduction of N_2: The Binding of Adenosine 5'-Triphosphate and Cyanide to the N_2-reducing System

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