Catalytic behavior of a polynuclear Mg-Mo complex and nitrogenase active site (FeMoco) isolated from the enzyme in reactions with C2H 2, N2, and CO: A comparative study

Article abstract of DOI:10.1007/s11172-006-0335-7

In order to identify common and distinctive features in the catalytic behavior of natural and artificial nitrogen-fixation clusters, the kinetics of the catalytic reduction of C₂H₂ in the presence of Mg-Mo-cluster (1) was investigated and compared with the kinetics of acetylene reduction catalyzed by the cluster FeMoco (2) isolated from the enzyme nitrogenase we studied previously. The reactions were conducted in the presence of Zn/Hg and Eu/Hg as reducing agents and PhSH and C₆F₅SH as proton donors, i.e., under the same conditions as had been used in the case of 2. Both polynuclear Mg-Mo-complex and the europium amalgam-reduced FeMoco have multiple interdependent binding sites for substrates and/or inhibitors. Carbon monoxide inhibits the acetylene reduction much less efficiently in systems with cluster 1 than in systems with cluster 2, although the type of inhibition is mixed in both systems: CO binds to multiple sites of the cluster and affects both C₂H₂ complexation to the reduced cluster and decomposition of the catalyst-substrate complex to give the products. Unlike isolated FeMoco, the Mg-Mo-cluster efficiently catalyzes the reduction of molecular nitrogen. The reaction is greatly inhibited by acetylene, while no inhibiting effect of N₂ is observed in acetylene reduction, as was found earlier for a system with the natural cluster as the catalyst. Springer Science+Business Media, Inc. 2006.

Full text of DOI:10.1007/s11172-006-0335-7

Russian Chemical Bulletin, International Edition, Vol. 55, No. 5, pp. 793—801, May, 2006
793
Catalytic behavior of a polynuclear Mg—Mo complex
and nitrogenase active site (FeMoco) isolated from the enzyme
in reactions with C2H2, N2, and CO: a comparative study
N. V. Bardina,ꢀ T. A. Bazhenova, G. N. Petrova, A. K. Shilova, and A. E. Shilov
Institute of Problems of Chemical Physics, Russian Academy of Sciences,
142432 Chernogolovka, Moscow Region, Russian Federation.
Fax: +7 (496) 524 4401. Eꢀmail: nadezhda@cat.icp.ac.ru
In order to identify common and distinctive features in the catalytic behavior of natural and
artificial nitrogenꢀfixation clusters, the kinetics of the catalytic reduction of C2H2 in the
presence of Mg—Moꢀcluster (1) was investigated and compared with the kinetics of acetylene
reduction catalyzed by the cluster FeMoco (2) isolated from the enzyme nitrogenase we
studied previously. The reactions were conducted in the presence of Zn/Hg and Eu/Hg as
reducing agents and PhSH and C6F5SH as proton donors, i.e., under the same conditions as
had been used in the case of 2. Both polynuclear Mg—Moꢀcomplex and the europium amalgamꢀ
reduced FeMoco have multiple interdependent binding sites for substrates and/or inhibitors.
Carbon monoxide inhibits the acetylene reduction much less efficiently in systems with clusꢀ
ter 1 than in systems with cluster 2, although the type of inhibition is mixed in both systems:
CO binds to multiple sites of the cluster and affects both C2H2 complexation to the reduced
cluster and decomposition of the catalyst—substrate complex to give the products. Unlike
isolated FeMoco, the Mg—Moꢀcluster efficiently catalyzes the reduction of molecular nitroꢀ
gen. The reaction is greatly inhibited by acetylene, while no inhibiting effect of N2 is observed
in acetylene reduction, as was found earlier for a system with the natural cluster as the catalyst.
Key words: biomimetics, biological nitrogen fixation, metal complex catalysis, metal clusꢀ
ter, active site, structural and functional models, mechanism, nitrogenase, acetylene
reduction.W
Previously,1 a nitrogenꢀreducing system based on polyꢀ
nuclear molybdenum complexes was discovered. Curꢀ
rently, this is the only known nonꢀenzyme system capable
of catalytic reduction of N2 at atmospheric pressure
and room temperature at rates comparable with nitroꢀ
genase. The reaction is carried out in methanol with a
minor water additive, which apparently serves as the proꢀ
ton donor in ammonia and/or hydrazine formation.
Sodium amalgam, europium amalgam or a cathode with
a specified potential equal to the Na/Hg potential can
serve as reducing agents.2 The molecular structure of
the Mgꢀcontaining molybdenum anionic cluster
{[Mg2Mo8O22(MeO)6(MeOH)4]2–[Mg(MeOH)6]2+}•
•6MeOH (1), which forms the active site of the system
upon reduction, has been determined3 by Xꢀray diffracꢀ
tion (Fig. 1).
The active site of the natural nitrogenꢀfixing enzyme
nitrogenase incorporates an octanuclear heterobimetallic
cluster FeMoco ((µ6ꢀN)MoFe7S9•homocitrate (2)) in
which nitrogen is bound and then reduced to ammonia.5
The chemical mechanism of the multielectron reduction
of nitrogenase substrates catalyzed by the cluster is unꢀ
known.
In order to elucidate the function of the cofactor and
the contribution made by the whole protein matrix and by
the amino acids located most closely to FeMoco to the
nitrogen reduction under mild conditions, we studied the
catalytic reactivity of the FeMoco cluster isolated from
the protein toward the reduction of nitrogenase substrates
in a nonprotein medium under conditions where elecꢀ
trons, protons, and the substrate were available to it.6 For
FeMoco as a catalyst of one nitrogenase reaction, namely,
C2H2 reduction without a protein, we found a number of
interesting features that are also characteristic of FeMoco
functioning in an enzyme system. In particular, the presꢀ
ence of a number of interdependent sites for substrate and
inhibitor coordination on the europium amalgamꢀreduced
FeMoco cluster was demonstrated and the type and paꢀ
The available data1—4 indicate that this compound is
reduced with retention of the cluster core, although,
strictly speaking, the structure of the complex active with
respect to N2 is unknown. The molecular mechanism of
N2 catalytic reduction with participation of this cluster is
also unknown.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 766—774, May, 2006.
1066ꢀ5285/06/5505ꢀ0793 © 2006 Springer Science+Business Media, Inc.

794
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
Bardina et al.
(chemically pure grade), molybdenum pentachloride (pure
grade), magnesium chloride (chemically pure grade), tributylꢀ
phosphine, mercury metal 99.9995% (Aldrich), argon (highꢀ
purity grade), and nitrogen (special purity grade), were used as
received.
Mo
Mg
O
C
Anhydrous methanol (pure grade, Reakhim) purified by reꢀ
fluxing with magnesium methoxide and distilled in an inert gas
flow was used as the solvent. Analytical solutions were prepared
using triply distilled water.
Acetylene (pure grade) was additionally purified by a known
procedure.6b,c Dideuterioacetylene was synthesized by the reacꢀ
tion of calcium carbide with heavy water (D2O, 99.8% deuteꢀ
rium) and purified in a similar way.6b Carbon monoxide was
synthesized by the reaction of sodium formate (Reakhim) with
concentrated sulfuric acid. Sodium, zinc, and europium amalꢀ
gams were prepared by dissolving the metals in mercury in an
inert atmosphere. The metal concentration was determined by a
published procedure.6а The prepared amalgams were stored unꢀ
der argon. All operations with substances sensitive to oxidation
were carried out under strictly anaerobic conditions using
Schlenk technique.
Complex 1 was synthesized from MoCl5 and MgCl2 in
methanol, as described previously.4 To prepare the active form
of the catalyst (designated in the Schemes as [Mg2Mo8]), the
complex was reduced by sodium amalgam as described previꢀ
ously.1
Fig. 1. Molecular structure of {[Mg2Mo8O22(MeO)6ꢀ
(MeOH)4]2–[Mg(MeOH)6]2+}•6MeOH (1).
Acetylene reduction. The experiments were carried out in a
specially designed thermostated flatꢀbottom glass vessel with a
magnetic stirrer, suitable for working with metal amalgams.8
The vessel was evacuated and filled with argon, then Eu/Hg
(0.5 mL) was introduced under argon into the side tumbler tube
and a solution (4—4.3 mL) containing complex 1 (5•10–6
mol L–1) and thiophenol (0.01 mol L–1) in MeOH was added
into the main vessel. After freezing of the liquid phase, the
reaction vessel was connected to a circulation unit and evacuꢀ
ated, then the liquid was unfrozen, and the vessel was filled with
a gas mixture. During the experiment, the gas phase was stirred
in the circulation unit and the liquid phase was stirred with a
magnetic stirrer under conditions that ensured the maximum
dispersion of the amalgam. The gas phase composition was varꢀ
ied depending on the task: for acetylene reduction, the vessel
was filled with an acetylene—argon mixture in different ratios
(to study the reaction stereospecificity, dideuterioacetylene
was used); to study the inhibitory effect of CO or N2,
a CO (or N2)—acetylene—argon mixture was used. In the exꢀ
periments dealing with the dependence of the reaction rate on
the catalyst concentration, the content of the complex in the
reaction mixture was varied in a range of (0.3—3)•10–5 mol L–1
at a constant acetylene pressure of 110 Torr (0.145 atm) and a
constant liquid phase volume.
rameters for inhibition of acetylene reduction with carꢀ
bon monoxide and molecular nitrogen were determined.
The binding constants of both substrates and inhibitors
with the FeMoco cluster reduced by europium amalgam
without a protein matrix were close to those found for the
nitrogenase Мꢀcenter (Мꢀcenter is the FeMoco cluster in
the MoFe protein).
Previously,7 we showed that, apart from N2 reduction,
the polynuclear Mg—Moꢀcomplex can catalyze the reꢀ
duction of C2H2, moreover, by much weaker reducing
agents than sodium amalgam, for example, by zinc or
europium amalgam, but in the presence of a stronger
proton donor, thiophenol.
In this work, we studied the kinetics of C2H2 reducꢀ
tion catalyzed by cluster 1 under the same conditions and
in the presence of the same reducing agents as have been
used previously6 to study the catalytic reactivity of FeMoco
isolated from the enzyme. Thus, it would be possible to
compare the mechanistic features of this reaction under
the same conditions in the presence of either synthetic
polynuclear complex 1 or the natural FeMoco cluster, in
order to find out how much they are similar as catalysts.
The reaction was monitored by taking gas samples from the
reaction vessel at regular intervals for chromatographic analysis
(see below). The resulting kinetic curves show the time depenꢀ
dence of the amount of the product formed. Using the initial
sections of the kinetic curves, the initial steadyꢀstate reaction
rate (W ), i.e., the amount of the reaction product formed per
unit time, was calculated.
Experimental
The commercial chemicals: tetraꢀNꢀbutylammonium broꢀ
mide, thiophenol (Fluka), pentafluorothiophenol (Sigma), phosꢀ
phatidyl choline (Kharkov, Bacterial Preparation Plant), soꢀ
dium hydroxide (Chemapol), anhydrous magnesium sulfate
Nitrogen reduction with europium amalgam. In the study of
nitrogen reduction with europium amalgam in the presence of
thiophenol or pentafluorothiophenol, the vessel was filled with
the liquid phase similarly to acetylene reduction experiments

Mg—MoꢀCluster and FeMoco catalytic activity
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
795
and then filled with nitrogen up to atmospheric pressure, and
the reaction was carried out for 30 min at 20 °C. After compleꢀ
tion of the reaction, the solution was analyzed for hydrazine and
ammonia (see below).
W•107/mol min–1
1
1.6
Nitrogen reduction with sodium amalgam. Inhibition. The reꢀ
duction of nitrogen was carried out in a vessel similar to that
used in acetylene reduction experiments but in this case, a samꢀ
pling capillary was inserted in the vessel to determine the hydraꢀ
zine and ammonia contents.8 The vessel was preꢀevacuated and
sodium amalgam (C = 1.07 mol L–1) (0.7 mL) was introduced in
a nitrogen flow into the side tumbler tube, while the main
vessel was filled with methanol (8 mL), a solution of NaOMe
(4.6 mol L–1) in methanol (0.01 mL), a solution of phosphatidyl
choline (0.13 mol L–1) in ethanol (0.02 mL), tributylphosphine
(4.013 mol L–1, 0.01 mL), and complex 1 (0.015 mL) with a
concentration of 4.9•10–3 mol L–1. After addition of the reacꢀ
tants, the vessel was filled with an N2—Ar mixture in nitrogen
reduction experiments and with an N2—C2H2—Ar mixture when
acetylene inhibition of nitrogen reduction was studied.
Experiments on electroreduction of acetylene and determinaꢀ
tion of the reaction activation energy were carried out in the
potentiostatic mode with a zinc amalgam working electrode by a
published procedure.9 All measurements at different temperaꢀ
tures were carried out at the same reactant concentrations and
other parameters; therefore, the activation energies were calcuꢀ
lated by the Arrhenius equation using reaction rates rather than
rate constants.
1.2
2
0.8
0.4
0
0.5
1.0
1.5
[Mg2Mo8]•105/mol L–1
Fig. 2. Initial rates of ethylene (1) and ethane (2) formation vs.
volume concentration of the catalyst upon acetylene reduction.
Reaction conditions: Eu/Hg (0.35 mol L–1) 0.5 mL; [PhSH] =
0.003 mol L–1; MeOH as the solvent (4.2 mL); [Mg2Mo8] as the
catalyst; 21 °C; pC H = 110 Torr.
2
2
to complete surface coverage of the given amount of amalꢀ
gam with the reduced cluster at a constant stirring rate.
Previously,6b it has been shown for a system involving the
FeMoco cluster (2) that the dependences of the ethylene
and ethane formation rates on the volume concentration
of the catalyst are described by an equation similar to the
adsorption isotherm equation that reflects the change in
the catalyst surface concentration upon an increase in its
volume concentration. This indicates that the reactions
giving the reduction products occur on the amalgam surꢀ
face without migration of the catalyst—substrate complex
to the solution bulk in both systems. This pattern of the
process is additionally confirmed by electrochemical exꢀ
periments, which show (see below) that the electron transꢀ
fer from the amalgam to the catalytic cluster adsorbed on
its surface is the rateꢀlimiting step.
Analytical procedures. The molybdenum concentration was
determined by spectrophotometry in the thiocyanate complex
(λ = 460 nm).10 The magnesium concentration was determined
by titration with Trilon B in an ammonia buffer solution (рH 10)
in the presence of Chromogen Black EТꢀ00 as an indicator.11
The concentration of hydrazine was determined by specꢀ
trophotometry as
ε
a colored compound (dihydrazone,
–1
max(458 nm) = 7•104 L mol
cm–1) via the reaction with
pꢀdimethylaminobenzaldehyde in an acid medium.12 The conꢀ
centration of ammonia was determined by the indophenol
method modified as described previously.13
The gaseous reaction products, viz., ethylene, ethane, and
methane, were analyzed by gas chromatography on a Biochrom
chromatograph (column filled with activated alumina
(0.25—0.5 mm), column temperature 90 °C, argon as the carrier
gas, flame ionization detector).
W•107/mol min–1
Absorption spectra were recorded on a Hewlett—Packard
8451A Diode Array Spectrophotometer. IR spectra were meaꢀ
sured on a Perkin—Elmer Spectrum Bx2 IR spectrometer in a
range of 4000—400 cm–1 with a 2 cm–1 resolution. The spectra
were recorded using a sealed KBr cell for gaseous samples. The
gaseous products of the catalytic reduction of C2D2 were sampled
using a Tepler pump from the reaction vessel cooled to –93 °C
by an ethanol bath.
1
3.5
3.0
2.5
2.0
1.5
1.0
0.5
2
Results and Discussion
Dependence on the catalyst concentration. The plot for
the ethylene and ethane formation rates vs. the volume
concentration of catalyst 1 in the reaction solution is
shown in Fig. 2. At Mg2Mo8 concentration >5•10–6
mol L–1, an apparent zero order is observed caused by the
saturation effect; probably, this concentration corresponds
0
100
200
300
400
pC H2/Torr
2
Fig. 3. Rates of ethylene (1) and ethane (2) formation vs. subꢀ
strate pressure in the reduction of C2H2. Reaction conditions:
Eu/Hg 0.5 mL; [PhSH] = 0.01 mol L–1; MeOH as the solvent
(4.2 mL); [1] = 5•10–6 mol L–1; C2H2; 21 °C.

796
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
Bardina et al.
W•107/mol min–1
0.8
Dependence on the substrate concentration. The curves
1
2
for ethylene and ethane formation rates over a wide range
of C2H2 pressures in the gas phase above the reaction
solution are presented in Fig. 3.
The products of C2H2 reduction are formed in parallel.
The pattern of the dependence (gradual increase in the
reaction rate without smoothing down) suggests a nonꢀ
Michaelis reaction mechanism,14 involving activation of
the reaction with excess substrate due to the formation of
ternary complex 3 (catalyst—substrate—substrate), which
differs in reactivity from the catalyst—substrate complex 4
(Scheme 1).
0.6
0.4
0.2
0
5
10
15
20
pC H2/Torr
2
Fig. 4. Rates of ethylene (1) и ethane (2) formation in the
acetylene reduction in the C2H2 pressure range of 3—22 Torr in
straight coordinates. Reaction conditions: see Fig. 3.
Scheme 1
may be indicative of the existence of different binding
sites on the catalytic cluster, one giving predominantly
ethylene and the other, ethane. This difference is also
attributable to the fact that under equilibrium conditions,
the Km value is determined by the rate constants for not
only binding of the substrate molecule to the cluster but
also for all the subsequent steps, resulting in the reduction
products (i.e., electron and proton transfer). Meanwhile,
the numbers and rates of such steps en route to ethylene
and ethane are different.
Thus, the obtained data show that the reduced cluster
has several substrateꢀcoordinating sites with different
acetylene affinities. Apparently, one, the most active site
that operates at low acetylene pressures gives equal proꢀ
portions of ethane and ethylene; indeed, for acetylene
pressure of up to 7 Torr, the ethane fraction in the reacꢀ
tion products is ~50%. For a similar reaction in the sysꢀ
tem with catalyst 2, we have also observed the existence of
several C2H2 coordination and reduction sites with differꢀ
ent substrate affinities, the Km values found being close in
magnitude in the two systems.
i
where Ks is equilibrium dissociation constant of ternary comꢀ
plex 3.
The reaction rate is described by the equation:
,
(1)
Inhibition of acetylene reduction by carbon monoxide.
It is known16 that CO inhibits the reduction of all nitrogeꢀ
nase substrates except for H+ in the case of enzyme caꢀ
talysis. The inhibiting effect is due to the fact that CO,
being isoeletronic to N2 and C2H2, is bound to the clusꢀ
ter, which has been reduced during the catalytic cycle,
although CO itself is not reduced.
Data on the effect of CO on the rate of C2H2 reducꢀ
tion catalyzed by cluster 1 are presented in Fig. 5. It can
be seen that the rates of formation of both ethylene and
ethane decrease in the presence of CO. Treatment of the
kinetic data on the inhibition by CO in the Dixon and
Cornish—Bowden coordinates (Figs. 6 and 7) allows one
to determine the inhibition type and calculate the process
parameters.15b Figure 6 shows the dependence of the iniꢀ
tial steadyꢀstate rate for ethylene and ethane formation
on the CO pressure in the system involving cluster 1 in the
α > 1 and K is > αKm (see Ref. 15),
where W is the reaction rate, S is the substrate concentraꢀ
tion, Km is the Michaelis constant, Vmax is the maximum
rate attained under conditions of cluster saturation with
the substrate, α is the correction factor.
To find the Michaelis constant, which is a measure of
the substrate affinity for the catalyst, we made use of the
fact that at low concentrations of the substrate, the term
containing S2 in expression (1) can be neglected, and the
equation will be simplified to the Michaelis—Menten
hyperbola equation.15a Figure 4 presents the experimenꢀ
tal data for low acetylene pressures (3—22 Torr). The
Michaelis constants found for the ethylene and ethane
formation reactions in the acetylene concentration range
chosen were 0.0048 and 0.0023 atm C2H2, respectively.
The fact that Km values for the two products are different

Mg—MoꢀCluster and FeMoco catalytic activity
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
797
W•107/mol min–1
pC H •W(C H )–1/Torr mol–1 min
2
2
2
4
1.2
1
200
160
120
80
1.0
0.8
0.6
0.4
1
a
2
2
10
20
30
pCO/Torr
–Ki´
40
Fig. 5. Rates of ethylene (1) и ethane (2) formation in the
acetylene reduction catalyzed by the Mg—Moꢀcomplex
and inhibited by carbon monoxide. Reaction conditions:
Eu/Hg 0.5 mL; [PhSH] = 0.01 mol L–1; MeOH as the solvent
–20
0
20
40
pCO/Torr
pC H •W(C H )–1/Torr mol–1 min
2
2
2
6
(4.2 mL); [1] = 5•10–6 mol L–1; pC H2 = 107 Torr; CO; 21 °C.
2.8
2
1
2.4
2.0
1.6
1.2
0.8
0.4
W(C H )–1/mol–1 min
2
4
b
1
2
2.4
a
1.6
0.8
2
–Ki´
–Ki
–100
–20
0
20
40
pCO/Torr
–60
–20
0
20
pCO/Torr
Fig. 7. Carbon monoxide inhibition of C2H2 reduction to
C2H4 (a) and C2H6 (b) (data in the Cornish—Bowden coordiꢀ
–0.8
–1.6
nates): pC H = 50 (1), 107 Torr (2). Reaction conditions:
2
see Fig. 5. 2
rant. With this form of the plot, one can directly deterꢀ
mine the inhibition constant (Ki), which is the reciprocal
of the cluster affinity for the inhibitor CO molecule. The
Ki values for ethylene and ethane are equal (0.14 atm) and
the inhibition is of a mixed type. Since the intersection
point of the straight lines lies below the х axis showing the
inhibitor pressure, the contribution of nonꢀcompetitive
inhibition is greater than that of competitive inhibition,
i.e., Ki > Ki´, where
W(C H )–1/mol–1 min
2
6
1
3.0
1.8
0.6
b
2
–Ki
–100
,
–60
–20
20
pCO/Torr
–0.6
.
–1.8
The constant Ki´ for product formation was found
using the Cornish—Bowden method. The plots for of the
ratio of the substrate pressure to the reduction rate
Fig. 6. Carbon monoxide inhibition of C2H2 reduction to
C2H4 (a) and C2H6 (b) with europium amalgam catalyzed by
the Mg—Moꢀcluster (data in the Dixon coordinates): pC H
=
2
2
50 (1), 107 Torr (2). Reaction conditions: see Fig. 5.
(pC H /W ) vs. the inhibitor pressure (pCO) are shown in
2
Fig2. 7. The resulting constants proved to be different for
ethylene and ethane formation; this may imply that CO is
attached to the complex that already has a substrate molꢀ
Dixon coordinates. The straight lines related to two difꢀ
ferent substrate concentrations intersect in the third quadꢀ

798
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
Bardina et al.
Scheme 2
Ki = 0.14 atm CO, Ki´(C2H4) = 0.02 atm CO, Ki´(C2H6) = 0.04 atm CO, Ks ≤ Ki
ecule in the coordination sphere at different reduction
steps.
The catalytic transformation of acetylene into ethylꢀ
ene and ethane in the presence of CO in the gas phase in
the system involving 1 is shown in Scheme 2.
A comparison of the data obtained on the carbon monꢀ
oxide inhibition of the reduction of C2H2 for cluster 1 as
the catalyst with these data for the FeMoco catalyst6c
shows qualitatively similar pictures. In both cases, subꢀ
stantial inhibition is observed, which is different for the
formation of ethylene and ethane. Quantitatively, the inꢀ
hibiting effect of carbon monoxide is much higher for the
ditions where C2H2 is efficiently reduced. The Mg—Moꢀ
complex, unlike FeMoco extracted from the protein, effiꢀ
ciently catalyzes the reduction of N2 to hydrazine and
ammonia (see, for example, Ref. 13 and references cited
therein). It was of interest to determine the characteristics
of the dinitrogen complex formed at the first step of N2
activation to compare them with the results obtained for
the FeMoco catalyst. However, in the presence of the
Mg—Moꢀcomplex, we did not find any inhibiting effect
of nitrogen toward the C2H2 reduction even for very low
acetylene concentrations in the gas phase. However, the
reduction of N2 is inhibited by acetylene very efficiently.
The kinetics of the catalytic reduction of nitrogen cataꢀ
lyzed by the polynuclear Mg—Moꢀcomplex has been studꢀ
ied in detail (see, for example, Ref. 7). High rates of N2H4
and NH3 formation are observed in soꢀcalled complete
system where sodium amalgam is used as the reducing
agent and phosphatidylcholine and tributylphosphine are
present as the coꢀcatalysts. Under these conditions,
acetylene is a very good substrate with Km of about
3•10–4 atm C2H2 (found from inhibition data). The satuꢀ
ration of the reaction rate with respect to nitrogen is obꢀ
served at N2 pressures of several tens of atmospheres,
while the Кm value for nitrogen calculated from data on
acetylene inhibition of nitrogen reduction is 25 atm. It is
not surprising that acetylene inhibits efficiently the reꢀ
duction of nitrogen under these conditions. The plot for
the nitrogen reduction rate in the presence of minor
amounts of acetylene in the gas phase is presented in
Fig. 8. It can be seen that the inhibiting effect is of nonꢀ
competitive type (the straight lines intersect in the negaꢀ
tive region of the х axis showing the inhibitor pressure)
and Ki is equal to 0.009 atm C2H2. A comparison with
similar data17 for the nitrogenase system in vitro shows
that the relationships of the characteristics found for acetyꢀ
lene and nitrogen as substrates in the natural system and
in its best functional model are closely similar.
system containing cluster 2: for pCO = 0.5pC H2 the rate of
2
acetylene reduction decreased ninefold; for the same subꢀ
strate to inhibitor ratio in the system with cluster 1, the
rate of acetylene reduction decreased only twice.
Apparently, a mixed type of inhibition is involved in
both cases, in particular, CO is coordinated to several
sites of the cluster and affects both C2H2 coordination to
the reduced cluster and decomposition of the cataꢀ
lyst—substrate complex to give the products. No reducꢀ
tion of CO is observed in either of the cases.
Data on the effect of CO on the reduction of acetylene
catalyzed by cluster 1 confirm the conclusion drawn from
the study of the variation of the rate vs. substrate concenꢀ
tration in this system, namely, there are several interdeꢀ
pendent coordination sites for substrates and inhibitors
on the reduced catalytic Mg—Moꢀcluster. A similar feaꢀ
ture is observed for the FeMoco catalyst.6b,c
Inhibition of nitrogen reduction by acetylene in the sysꢀ
tem involving the synthetic Mg—Moꢀcluster. While invesꢀ
tigating the catalytic reduction of C2H2 catalyzed by
FeMoco, we found that molecular nitrogen inhibits comꢀ
petitively the reduction of C2H2 through the reversible
complexation with reduced cluster 2 (see Ref. 6c). In the
absence of protein, no subsequent protonation of the reꢀ
sulting nitrogen complex takes place, at least, under conꢀ

Mg—MoꢀCluster and FeMoco catalytic activity
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
799
–1
W(N H ) •10–6/mol–1 min
lnW
2
4
–2.4
a
2.0
1
2
–2.8
1.5
1.0
0.5
1
–3.2
–3.6
–Ki
2
0
–8
–4
4
8
pC H2/Torr
2
–4.0
3.25
lnW
3.30
3.35
3.40
T
–1•103/K–1
Fig. 8. Acetylene inhibition of N2 reduction in the complete
system at pN2 = 400 (1), 760 Torr (2) (data in the Dixon coordiꢀ
nates). Reaction conditions: Na/Hg (1.07 mol L–1), [Bu3P] =
–0.8
–1.2
–1.6
–2.0
5•10–3 mol L–1, [phosphatidyl choline] = 3•10–4 mol L–1
,
,
[NaOMe]
=
6•10–3 mol L–1
,
[1]= 8.3•10–6 mol L–1
b
[Bu4NBr] = 0.1 mol L–1
.
1
2
Electrochemical experiments and temperature deꢀ
pendence. Previously, for systems containing the
Mg—Moꢀcluster7,13 and FeMoco isolated from a proꢀ
tein9 as catalysts, the rate of C2H2 reduction was found to
depend exponentially on the specified potential of a zinc
amalgam cathode in the presence of proton donors such
as thiophenol or water. This implies that for both systems,
electron transfer from the cathode to the catalytic cluster
is the rateꢀlimiting step of the process. While investigating
the temperature dependences of acetylene reduction cataꢀ
lyzed by cluster 2 with PhSH as the proton donor, we
found9 that the effective activation energies for the forꢀ
mation of ethane and ethylene are different, Ea for the
formation of C2H6 being ~1.5 times lower than that for
C2H4 (Table 1). It was concluded that the structure and
properties of the natural catalytic cluster are favorable for
multielectron processes.
3.25
3.30
3.35
3.40
T
–1•103/K–1
Fig. 9. Temperature dependences of the ethylene (1) and
ethane (2) formation rates during the catalytic reduction of acetyꢀ
lene with the Mg—Moꢀcomplex in the presence of thiophenol (a)
and pentafluorothiophenol (b); Arrhenius coordinates. Reacꢀ
tion conditions: the specified potential of the zinc amalgam
cathode E = –1.3 V; MeOH as the solvent; the catholyte volume
6.5 mL; the catalyst [1] = 1.32•10–5 mol L–1; [PhSH] =
[C6F5SH] = 1.2•10–2 mol L–1; pC H2 = 100 Torr.
2
thiophenol (Fig. 9). For the system with cluster 2, an
increase in the proton donor acidity resulted in a change
in the rateꢀlimiting step, which was manifested as the lack
of dependence of the acetylene reduction rate on the
cathode potential in the presence of C6F5SH. Hence, the
system with the Mg—Moꢀcomplex depends to a lesser
extent on the acidity of the protonating agent. Having
calculated the effective activation energies (Ea) from
the Arrhenius equation, we found that with the
Mg—Moꢀcomplex as the catalyst, too, Ea for C2H6 forꢀ
mation is lower than Ea for C2H4 formation (see Table 1).
Thus, a common feature of these clusters is that they
catalyze multielectron processes. The absolute values of
Ea are markedly higher for the reaction in the presence of 2.
Note that the Ea values for 2 were obtained from experiꢀ
ments carried out at a potential of –1.7 V, while for the
system with cluster 1, at a potential of –1.3 V (vs. satuꢀ
rated silver chloride electrode Ag/AgCl/KClsat). Since a
Study of the temperature dependence of C2H2 reducꢀ
tion rate with cluster 1 as the catalyst showed that the
rates of ethylene and ethane formation increase exponenꢀ
tially with temperature rise in the range from 15 to 35 °C
not only in the presence of thiophenol, but also in the
presence of a more acidic proton donor, pentafluoroꢀ
Table 1. Activation energies for the formation of ethylene and
ethane in the catalytic systems containing clusters 1 and 2 in the
presence of thiophenol and pentafluorothiophenol
Catalyst
Reaction conditions
Еa/kcal mol–1
C2H4 C2H6
Reagent
–E/V
FeMoco12
PhSH
C6F5SH
PhSH
1.7
1.3
1.3
1.3
18.3 1.5
32
9.4 0.6
7.1 0.5
12.8
32
5.4 0.6
3.7 0.9
2
4
4
Mg—Moꢀ
complex
C6F5SH

800
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
Bardina et al.
T (%)
C2HD, and C2H2. This reaction occurs on the Eu/Hg
surface equally efficiently both in the presence and in the
absence of the catalytic cluster in solution, the exchange
of C2D2 with the PhSH or MeOH protons being faster
than the catalytic reduction of C2D2. The reduction of a
mixture of acetylenes yields a set of all possible ethylene
isotopes without predominant formation of either cisꢀ or
transꢀproduct (Fig. 10).
2
1
120
100
80
C2HD
T (%)
C2H2
transꢀ1,2ꢀC2H2D2
60
Some characteristics of the reduction of C2H2 and
nitrogen with europium amalgam in the presence of the
Mg—Moꢀcomplex and FeMoco were compared (Table 2).
It can be seen that the regular features of these reactions
in the systems involving the natural catalytic FeMoco
cluster and the synthetic Mg—Moꢀcomplex simulating
the function of this cluster are qualitatively similar in
many respects.
The most pronounced differences between these sysꢀ
tems are related to the reaction with molecular nitrogen.
In the presence of cluster 2, only reversible complexation
of nitrogen with the reduced cluster takes place, while
with cluster 1, N2 is reduced to hydrazine and ammonia.
In the latter case, we did not succeed in obtaining any
characteristics of the primary complexation of N2, beꢀ
cause no inhibition of the acetylene reduction by nitrogen
was observed in this system. Apparently, even under conꢀ
ditions far from saturation with nitrogen, the dinitrogen
complex formed is rapidly and irreversibly protonated to
give N2H4 and NH3.
Analysis of the data obtained on the comparative beꢀ
havior of the clusters suggests that it would be possible to
create a catalytic system with FeMoco without protein
that would perform the reduction of molecular nitrogen.
This requires creating a better contact of the catalytic
cluster with the surface of the electron donor (amalgam or
cathode), as has been done previously in the system with
the Mg—Moꢀcluster in a lipid film,8 by selecting approꢀ
priate surfactants or using electrodes with special coatings
capable of strong adsorption of FeMoco.
117.1
117.0
cisꢀ1,2ꢀC2H2D2
ν/см–1
40
C2H4
1000
900
920
800
C2D2
700
600
ν/cm–1
Fig. 10. IR spectra of the initial acetylene C2D2 (1) and the
products of C2D2 reduction catalyzed by the Mg—Moꢀcomꢀ
plex (2); the inset shows the enlarged fragment of spectrum 2
corresponding to the reduction products. Reaction conditions:
Eu/Hg (0.5 mL); [PhSH] = 0.01 mol L–1; MeOH as the solvent
(4.2 mL); [1] = 5•10–6 mol L–1; pC D2 = 120 Torr; 21 °C.
2
decrease in the potential, with other factors remaining
invariable, entails a decrease in the activation energy for
those systems in which electron transfer is the rateꢀlimitꢀ
ing step, the observed difference indicates that a better
spatial organization and a stronger contact with the amalꢀ
gam (reducing agent) are attained in the system with the
Mg—Moꢀcomplex. This facilitates the electron transfer
and decreases the activation energy.
Reaction stereospecificity. In order to elucidate the
type of binding of the C2H2 molecule to the cluster and
the mechanism of protonation of the coordinated acetyꢀ
lene, we attempted to study the stereospecificity of ethylꢀ
ene formation from C2D2 upon protonation with PhSH
or MeOH using FT IR spectroscopy. We did not obtain
unambiguous information, as under experimental condiꢀ
tions, C2D2 underwent fast and almost quantitative H/D
exchange on europium amalgam to give a mixture of C2D2,
Table 2. Comparison of some parameters of the reduction of nitrogenase substrates catalyzed by FeMoco isolated from
the protein and by the synthetic Mg—Moꢀcomplex
Catalyst
Substrate
Acetylene
COa
Nitrogen
Km•103
/atm C2H2
pb (%)
transꢀC2H2D2
Ki•103
/atm C2H2
Ic
Km
IId
Ki
C2H4
C2H6
C2H2
C2H4
C2H6
atm N2
FeMoco without protein
Mg—Moꢀcomplex
6
4.8
6
2.3
22
47
11
—
3
140
6
140
—
~25
0.49
—
a
A mixed inhibition mechanism is involved for both catalysts.
Fraction.
b
c I is the reduction of nitrogen.
d II is acetylene inhibition of the reduction of nitrogen.

Mg—MoꢀCluster and FeMoco catalytic activity
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 5, May, 2006
801
Petrova, A. K. Shilova, N. I. Shuvalova, and A. E. Shilov,
Inorg. Chim. Acta, 1998, 270, 221.
This work was supported by the Russian Foundation
for Basic Research (Project No. 04ꢀ03ꢀ32635).
8. L. P. Didenko, O. K. Gavrilina, E. E. Yablonskaya, A. K.
Shilova, and A. E. Shilov, Nouv. J. Chim., 1983, 7, 605.
9. T. A. Bazhenova, N. V. Bardina, G. N. Petrova, and M. A.
Borovinskaya, Izv. Akad. Nauk, Ser. Khim., 2004, 1583 [Russ.
Chem. Bull., Int. Ed., 2004, 53, 1646].
10. A. I. Busev, Analiticheskaya khimiya molibdena [Analytical
Chemistry of Molybdenum], Izdꢀvo Akad. Nauk SSSR, Mosꢀ
cow, 1962, 205 pp. (in Russian).
11. E. V. Alekseevskii, R. K. Gol´ts, and A. P. Musakich,
Kolichestvennyi analiz [Quantitative Analysis], Goskhimizdat,
Leningrad, 1957, 391 pp. (in Russian).
12. M. Perez and A. Petit, Bull. Soc. Chim. Fr., 1947, 122.
13. T. A. Bazhenova, M. A. Bazhenova, G. N. Petrova, A. K.
Shilova, and A. E. Shilov, Izv. Akad. Nauk, Ser. Khim.,
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prakticheskii kurs [Biokinetics. Practical Course], FairꢀPress,
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Received November 12 , 2005;
in revised form February 6, 2006