ORIGINAL PAPER

The mechanism of hydrogen uptake in [NiFe] hydrogenase:first-principles molecular dynamics investigationof a model compound

Sara Furlan • Giovanni La Penna

Received: 18 May 2011 / Accepted: 15 August 2011 / Published online: 3 September 2011

� SBIC 2011

Abstract The recent discovery of a model compounds of

[NiFe] hydrogenase that catalyzes the heterolytic cleavage

of the H2 molecule into a proton and a stable hydride in

water solution under room conditions opened up the pos-

sibility to understand the mechanism of H2 uptake by this

peculiar class of enzymes. The simplest model compound

belongs to the class of NiRu bimetallic cationic complexes

mimicking, in water solution and at room conditions, the

hydrogenase active site. By using first-principles molecular

dynamics computer simulations, in the Car–Parrinello

scheme, we investigated models including the water sol-

vent and nitrate counterions. Several simulations, starting

from different initial configurations, provided information

on the first step of the H2 cleavage: (1) the pathway of H2

approach towards the active site; (2) the role of the

ruthenium-bonded water molecule in providing a base that

extracts the proton from the activated H2 molecule; (3) the

minor role of Ni in activating the H2 molecule and its role

in stabilizing the hydride produced.

Keywords Hydrogenase � Hydrogen uptake � Computer

simulations � First-principles molecular dynamics

Introduction

[NiFe] hydrogenase is an enzyme, expressed in microor-

ganisms adapted to anaerobic conditions, which catalyzes

the cleavage of the H–H bond of the H2 molecule in water

solution at room temperature and pressure [1]. In particular

conditions the enzyme catalyzes the production of H2 by

reduction of protons using electron carriers produced by

fermentative processes, thus behaving like the peculiar

[FeFe] hydrogenase enzyme in the photoproduction of H2

by cyanobacteria and unicellular algae. These remarkable

properties, which may have a great impact in developing

sustainable H2 production and usage, are possible because

of the presence of metal ions in the enzyme active sites,

namely, Fe and Ni. The mechanism for the reaction,

H2 !MðLÞ

2Hþ þ 2e�; ð1Þ

where M(L) is a generic metal complex, is the subject of

intensive investigations [1–3] and several mechanistic

hypothesis have been proposed, with considerable help

provided by density functional theory (DFT) calculations

[1, 4–7]. Moreover, hydrogenases inspired a series of

supramolecular assemblies for Pt-free catalysis of

H2 ? H? interconversion [8, 9], the design of which

deserves the understanding of the molecular mechanism.

To provide a simple frame for understanding the

mechanism of action of [NiFe] hydrogenase, several model

compounds were synthesized and characterized [10–12]

with the aim of isolating the active site for H2 cleavage

from the hydrogenase protein matrix. Among these, the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-011-0838-z) contains supplementarymaterial, which is available to authorized users.

S. Furlan

LCC-Laboratory of Coordination Chemistry,

CNRS-National Center for Scientific Research,

205 route de Narbonne,

31077 Toulouse, France

G. La Penna (&)

CNR-National Research Council of Italy,

ICCOM-Institute for Chemistry of Organo-metallic Compounds,

Via Madonna del Piano 10,

50019 Sesto Fiorentino,

Florence, Italy

e-mail: glapenna@iccom.cnr.it

123

J Biol Inorg Chem (2012) 17:149–164

DOI 10.1007/s00775-011-0838-z

class of NiRu bimetallic compounds has been proposed as

the best candidate for efficient catalysis in terms of chem-

ical stability and turnover frequency [13]. One of the sim-

plest model compounds of this class fully mimicking the

enzyme is that recently discussed in [14, 15] and first

reported in [16]. In [16], the X-ray structure of a simple

Ni(L)Ru(L0) bimetallic water soluble complex (complex 1,

hereafter), able to form a stable hydride upon H2 addition to

a water solution under room conditions, was reported. The

ligands L and L0 are N,N0-dimethyl-N,N0-bis(2-mercapto-

ethyl)-1,3-propanediamine and g6-C6Me6 (1,2,3,4,5,6-

methylbenzene), respectively. The reaction, occurring by

bubbling H2 gas in a water solution of 1 at T = 293 K and

P = 1 bar, is

1ðH2OÞ2þ þ H2 ! 1ðHÞþ þ H3Oþ: ð2Þ

The replacement of Fe, present in the [NiFe]

hydrogenase enzyme, with Ru has been done because of

the higher stability of Ru compared with Fe in

electrochemical studies [12, 13]. Both Ni and Ru ions

have formal oxidation state II in the reactant complex, the

complex having ?2 charge. A water molecule is bonded to

Ru in the crystallized Ni(L)Ru(L0)H2O(NO3)2 salt, and

mass spectrometry and NMR data show that the water

molecule is bonded to the complex also in water solution.

In the same study, the structure of the hydride product was

investigated by neutron diffraction, showing the presence

of the hydride bridging Ru and Ni on the opposite side of

the two S atoms in L.

The compound investigated in this [NiFe] hydrogenase

biomimetic complex is shown in Fig. 1.

At the basis of the formation of the bridging hydride

intermediate there is the extraction of the proton from a

generic metal-activated H2 molecule. Despite several

computational models having contributed significantly to

the understanding of this mechanism [7], a complete model

for the mechanism of the heterolytic H2 cleavage must

include both the pathway for the H2 activation and the

approach of a base (most likely a water molecule or a

portion of the ligand), this latter step being necessary to

extract the proton from the activated H2 molecule [10, 15].

Several hypotheses concern both the location of the

activated H2 molecule and the nature of the base involved

in the [NiFe] hydrogenase active site. For the NiRu model

compound here investigated, these hypotheses are sum-

marized in Fig. 2.

In mechanism A (Fig. 2a, REP pathway hereafter), the

water molecule in the coordination sphere of Ru is replaced

(therefore the REP acronym) by H2 and the following H2

polarization and heterolytic cleavage occur on the Ru site,

with proton donation to a water molecule in the bulk [6]. In

mechanism B (Fig. 2b, Ru pathway hereafter), both H2 and

H2O molecules are coordinated to Ru and the H2 polariza-

tion and cleavage are assisted by the Ru-bonded water

molecule. Water is ejected from the Ru site when the

hydride is formed [15]. In mechanism C (Fig. 2c, Ni path-

way hereafter), the H2 polarization occurs when H2 interacts

axially with Ni and the H2 cleavage is assisted by the Ru-

bonded water [15]. This mechanism is also suggested by the

larger H2 accessibility of the Ni site in [NiFe] hydrogenase

[17]. Noticeably, the characterization of species 2 and 4 as

reaction intermediates has been provided by DFT calcula-

tions for similar NiRu model compounds [7, 18].

In the model compound, the persistence of the water

molecule bonded to Ru in water solution indicated a pos-

sible role for this metal-bonded water molecule as a weak

base localized close to the site of H2 activation [15]. The

competition between this possible base and the other

groups present in the system (metal-bonded thiolate groups

and water molecules of the solvent) deserves suitable

modeling techniques that include the water solvent

explicitly.

In this work the first stage of the H2 heterolytic cleavage

by complex 1 was investigated by means of first-principles

molecular dynamics simulations performed in the Car–

Parrinello scheme [19, 20], together with structural in

vacuo relaxation of several configurations identified by

experiments. All the calculations performed here were

within a DFT approach for the ground-state electronic

structure [21]. Previous first-principles molecular dynamics

calculations of Ru complexes in explicit solvents [22–24]

showed the ability of this computational method to provide

information complementary to DFT studies of reaction

mechanisms. The calculations reported here allow (1) the

identification of possible pathways for the approach of H2

towards the Ru and Ni centers of the reactant bimetallic

complex; (2) the identification of the Ru-bonded water as

an essential mean for the proton extraction from the metal-

activated H2 molecule; (3) the final pathway towards the

bridging hydride by means of water and counterion release,

due to the lower positive charge of the favored H–Ru

hydride intermediate.Fig. 1 Compound 1, i.e., the reactant in the reaction in Eq. 2 before

H2 addition

150 J Biol Inorg Chem (2012) 17:149–164

123

Despite the model compound investigated here being

chemically different from the [NiFe] hydrogenase active

site, it preserves the peculiar catalytic activity of this family

of enzymes, as reported in the aforementioned experimental

studies. The computational study reported here shows how

the pathway from reactant to product is influenced by envi-

ronmental conditions, particularly by the density of water

molecules and counterions around the bimetallic cation. For

the first time, the role of these variables is accounted for in a

computational study of a compound of this class.

Methods

Car–Parrinello molecular dynamics (CP-MD) simulations

[19] were performed on systems in the range of 218–287

atoms. All the systems consist of complex 1 (21C, 40H, O,

2N, 2S, Ni, Ru, giving a total of 68 atoms), with, depending

on the case, additional counterions (two NO3- anions), the

Ru-bonded water molecule replaced by H2, several added

H2 molecules (in the range of one to four molecules), and a

maximum of 67 water molecules mimicking the bulk

solvent.

The parallel version of the Quantum-Espresso package

[25], which incorporates Vanderbilt ultrasoft pseudopo-

tentials [26] and the Perdew–Burke–Ernzerhof exchange–

correlation functional [27], was used in all CP-MD simu-

lations. Electronic wave functions were expanded in plane

waves up to an energy cutoff of 25 Ry, and a 250-Ry

energy cutoff was used for the expansion of the augmented

charge density in the proximity of the atoms, as required in

the ultrasoft pseudopotential scheme. The choice of ultra-

soft pseudopotentials is dictated by the fact that heavy

atoms would have required a large energy cutoff if standard

norm-conserving pseudopotentials had been employed

[26, 28]. A critical discussion on the validity of these DFT

approximations for the type of compounds analyzed in this

work is reported in the electronic supplementary material.

However, mainly because of the length of the trajectories,

the simulations reported here must be regarded as samples

that aim at discovering which of the alternative pathways

displayed in Fig. 2 are assisted by the molecular environ-

ment. For a more quantitative description of the reaction,

refined approaches are required, including, for instance, the

following advances in the context of first-principles

molecular dynamics (see [20] and the electronic supple-

mentary material): (1) a different DFT setup for water–

water [29] and then water–solute interactions; (2) suitable

quantum corrections for treating the movement of light

atoms such as H [30, 31]; (3) non-ground-state and mul-

tideterminant approaches, which are particularly important

when many transition metal ions allow multiple spin states

Fig. 2 The proposed mechanisms for the reaction in Eq. 2. See the text for details

J Biol Inorg Chem (2012) 17:149–164 151

123

[32]. For example, the correction in point 2 is shown to

contribute significantly to configurational fluctuations even

at room temperature, thus extending the exploration of the

ground-state potential energy surface especially in the case

where H–H bond fluctuations are involved and coupled

with O–H bond fluctuations. These corrections are extre-

mely computationally demanding and are beyond the

qualitative scope of this study, but they can be introduced

in the calculation of kinetic constants once the more likely

reaction pathway has been addressed.

To minimize finite volume effects, periodic boundary

conditions are imposed on the system. The CP-MD cal-

culations were performed under spin-restricted conditions

in all cases where the spin state has Sz = 0. In the Sz [ 0

cases, the DFT local spin density approximation was used.

Simulations were conducted according to the following

general protocol consisting of the three sequential steps: (1)

minimization of electronic energy with fixed atomic posi-

tions; (2) minimization of total energy as a function of both

atomic and electronic degrees of freedom; (3) a series of

sequential CP-MD simulations of about 0.2–0.3 ps, each at

fixed increasing atomic temperatures (in steps of

50–100 K, up to 300 K) with the temperature held fixed by

a Nose–Hoover thermostat [33].

The energy minimization of steps 1 and 2 was per-

formed via damped CP-MD, with a damping frequency for

all the degrees of freedom of 1/(10 dt) and with dt, the time

step, of 0.12 fs used for all the CP-MD simulations in this

work. The number of minimization time steps was about

1,000, depending on the system size. It must be noted that

this minimization does not yield a minimum in the total

energy, as expected in geometry optimizations, because of

the large number of degrees of freedom involved. Steps 1

and 2 are required to begin the following T [ 0 CP-MD

simulation with atomic velocities of low magnitude: in all

cases the maximal initial velocity for any atom was smaller

than 0.003 A/fs. The equilibration procedure described in

step 3 is necessary to slowly reach room temperature and

thus avoid temperature oscillations that may affect in an

uncontrolled way the approach of electrons to their ground

state. The velocity-Verlet algorithm [34] for integrating the

Car–Parrinello equations of motion was used with a time

step of 0.12 fs. The times spent during the different phases

of the simulations are summarized in Table 1, together

with details concerning the model construction.

The simulation times used in the applications reported in

this work are not long enough to provide statistical results.

The CP-MD method was used here to test the availability

of H2 cleavage pathways and their consistency with real-

istic thermal fluctuations mediated by solvent molecules.

By the use of dynamical algorithms, such as the molecular

dynamics method, energy barriers between local energy

Table 1 Summary of Car–Parrinello molecular dynamics (CP-MD) simulation stages and conditions

Model Atoms Setup Thermalization Volume

REP pathway (Fig. 2a)

M1 276 X-ray ? 2 NO3- ? 67 H2O 0.69 (50) ? 0.29 (150) ? 0.38 (300) 2.77

M2 276 End of M1, then at P = 10 bar 0.24 (50) ? 0.56 (100) ? 0.24 (200) ? 0.24 (300) 2.35

M3 276 Start of M1, H2 manually cleaved 0.36 (50) ? 0.36 (100) ? 0.36 (200) ? 0.73 (300) 2.77

M4 285 End of M2, then 3 H2 and H2O inserted

and 1.28 ps, P = 10 bar (Sz = 1)

0.73 (50) ? 1.06 (100) ? 0.73 (200) ? 0.73 (300) 1.93

M5 284 End of M2, then 4 H2 inserted and

1.28 ps, P = 10 bar

0.36 (50) ? 0.36 (ox. 50) ? 0.36 (ox. 100) 0.36 (50) ? 0.36

(100) ? 0.36 (200) ? 0.73 (300)

1.85

Ru pathway (Fig. 2b)

M6 285 End of M4, H2 exchanged with H2O in

the Ru site

0.36 (50) ? 0.36 (100) ? 0.36 (200) ? 0.73 (300) 1.93

M7 287 End of M6, H2 inserted close to Ru 0.23 (50) ? 0.36 (ox. 50) ?0.36 (50) ? 0.36 (100) ? 0.36

(200) ? 0.36 (300)

1.93

M7a 287 End of M7, P = 1 bar 0.24 (50) ? 0.48 (100) ? 0.48 (200) 0.73 (300) 1.88

M7b 287 End of M7a, same box as M3 0.36 (50) ? 0.36 (100) ? 0.30 (200) 1.45 (300) 2.77

Ni pathway (Fig. 2c)

M8 285 End of M4, H2 moved close to Ni 0.36 (50) ? 0.36 (100) ? 0.36 (200) ? 0.73 (300) 1.93

M9 285 Start of M8 0.36 (50) ? 0.36 (ox. 50) ?0.36 (ox. 100) ? 0.36 (ox. 200) ? 0.73 (ox.

300) ?0.36 (50) ? 0.36 (100) ? 0.36 (200) ? 1.09 (300)

1.93

M10 285 M8 after 0.36 ps ox. at T = 50 K then

reduced (Sz = 1)

0.36 (50) ? 0.36 (100) ? 0.36 (200) ? 0.36 (300) 1.93

Simulation times are in picoseconds; temperature (within parentheses) and external pressure are in kelvins and bars, respectively; volume is in

nanometers3. The ox. label indicates the deposition of ?2 charge (two-electron oxidation) in the simulation box

152 J Biol Inorg Chem (2012) 17:149–164

123

minima achieved by geometry optimization of in vacuo

models can be overtaken. Nevertheless, larger statistics

and/or suitable methods [35] are required for reliable

estimates of transition rates along the pathways identified.

The initial atomic configuration for cationic solute 2,

used as the building block for the first solvated model

investigated, was the X-ray structure [16] of species 1 (see

Fig. 2). All water molecules and counterions in the crystal

structure were discarded and species 1 was kept. The

Ru-bonded water molecule of 1 was replaced by H2 (with a

H–H distance of 0.74 A and with H–Ru distances of

1.7 A). This structure was placed in a supercell of

1.49 9 1.33 9 1.40 nm3 filled with water molecules, these

latter molecules being in a configuration taken from a

Monte Carlo simulation of liquid water under room con-

ditions [36]. All water molecules with a H–X distance less

than 1.5 A and an O–X distance less than 2.5 A, where X is

any atom of the initial structure, were discarded. The size

of the supercell was chosen as a compromise between

computational resources and the extent of water solvation.

The number of final water molecules added was 69. Two

nitrate counterions (present in the experiments performed

in water solution), with standard initial geometry, were

manually placed on the two different sides of the cationic

complex, replacing two water molecules in the supercell,

thus neutralizing the system. The initial configuration for

the starting simulation of this work is displayed in Fig. 3.

Additional H2 and H2O molecules were added in several

cases (see below and Table 1) manually, according to the

design of each model. All the initial manipulations were

performed with the program VMD [37].

To sample configurations where water molecules are

close to the solute species, the water density around the ions

was, in several cases, increased by performing simulations

at a constant pressure of 10 bar, using the Rahman–Parri-

nello algorithm [38] in conjunction with a constant tem-

perature bath. This step allows the adjustment of cell size

and shape. The cell remained almost orthorhombic, but the

use of P = 10 bar implied a significant volume reduction.

In simulation M2, at T = 300 K and P = 10 bar, the vol-

ume of the supercell was 2.35 ± 0.03 nm3 compared with

the M1 volume of 2.77 nm3. The equilibrium volume of the

supercell decreased when additional reactant H2 molecules

were included: the minimal cell volume of 1.85 nm3 was

obtained with simulation M5, i.e., model M2 with four

additional H2 molecules.

Several configurations obtained by CP-MD simulations

were analyzed in vacuo for comparison of energies and

geometries. In these cases, the energy of the models was

minimized by using the Broyden–Fletcher–Goldfarb–

Shanno method, with a threshold of 10-3 Ry/bohr for the

maximal modulus of any atomic force. We refer to this

latter minimization as ‘‘relaxation,’’ and the final configu-

rations obtained by this procedure as ‘‘relaxed’’ configu-

rations. This procedure is possible only for in vacuo

models. In the case of relaxation, the size of the supercell is

larger than in the CP-MD simulations to achieve better

accuracy in total energy. The supercell is always cubic with

an edge of 1.8 nm. The Makov–Payne correction [39],

accounting for the energy contribution of collecting the

charge in the given periodic supercell, is always included

in the reported energies. To account for intramolecular

dispersive forces, which are disregarded in the DFT

approach described above, one reported approach, the

DFT-D approximation [40], was used in the relaxation (see

the electronic supplementary material for details).

In the analysis of the electronic ground state, the mea-

sure of valence charge localized on atoms was performed

with the quantum theory of atoms in molecules [41], using

the algorithms reported in [42, 43].

The radial distribution functions (RDF) displayed in

‘‘Results’’ are, as usual, the number of pairs representing

the given distance in the simulation, divided by the same

number in the ideal gas with the same density of particles.

To roughly estimate the water solvent effects on the

computed in vacuo energy differences upon proton

extraction, the experimental absolute solvation free energy

Fig. 3 The initial configuration for Car–Parrinello molecular dynam-

ics simulation M1, including the g-H2 adduct to complex 1 shown in

Fig. 1 (species 2 in Fig. 2a), two NO3- anions, 67 water molecules

(represented as transparent objects). The sides of the simulation box

are represented as white sticks, with dimensions 14.9, 13.3, and

14.0 A, respectively. The color scheme is as follows: Ru is green, Ni

orange, C gray, H white, O red, N blue, and S yellow. The sizes of the

atoms and bonds are arbitrary. The VMD program [37] was used for

all the molecular drawings in this work

J Biol Inorg Chem (2012) 17:149–164 153

123

of the proton was added to the energy of the in vacuo

products, this quantity being measured within -1,050 and

-1,150 kJ/mol [44]. We added -1,107 kJ/mol, this

quantity often being used in quantum chemistry modeling

of similar reactions [45]. The solvation free energies for the

other species involved, such as H2, can be safely disre-

garded, these quantities being within the experimental error

for a proton (about 100 kJ/mol).

All calculations were performed with 32–128 parallel

computational tasks on the Juropa supercomputer of the

Julich Supercomputing Centre (Germany), and calculations

on truncated models were performed on the IBM SP6

supercomputer at Cineca (Bologna, Italy) and on a local

Linux cluster of CNR-ICCOM (Italy).

The advantage of the CP-MD technique, compared with

other DFT-based calculations, is the possibility to include

in the models a statistical sample of liquid water with

counterions (in this case nitrate anions) surrounding the

cationic complex. The inclusion of such a small sample of

the cationic environment allows one to investigate the

possibility of proton exchange with solvent water mole-

cules, eventually competing with proton exchange between

groups in the complex.

Results

The three possible pathways for heterolytic H2 cleavage,

the REP, Ru, and Ni pathways, are exploited by simulating

the behavior of configurations close to the hypothetical

intermediates displayed in Fig. 2. The results are summa-

rized in Table 2, with references to the following discus-

sion and to species identified in Fig. 2.

Energies and structures of relaxed configurations

In Fig. 4, the energies of reactant species 1 (assumed as the

energy reference) and REP mechanism intermediate 2 and

product 5 (see Fig. 2a) are displayed. The energies were

computed in the configurations relaxed in vacuo and in the

spin configurations indicated. Estimates of dispersive

contributions and of solvation free energy for H? are

included (see ‘‘Methods’’ for details).

The diagram shows that the product 5, with the octa-

hedral Ni coordination observed in experiments and in a

high-spin configuration, is the lowest-energy species in

the water solvent, whereas the H2 adduct 2 is the lowest-

energy species in vacuo. The hydride product 5 is also

stable in the low-spin configuration and in a square-

pyramidal coordination, i.e., with a marginal further sta-

bilization provided by the octahedral Ni coordination. The

inclusion of dispersive interactions does not change the

optimized geometries (see the electronic supplementary

material for details), whereas, as expected, it provides a

positive energy contribution for the formation of species 2

because of the steric hindrance of the Ru-associated H2

molecule.

These data show that the role of the water solvent, and

possibly of counterions, is important in driving the ener-

getic contribution to the thermodynamic of transformation

of complex 1 into the hydride 5. These contributions are by

far dominant also in the accuracy of the method, with the

errors in the experimental and computational methods for

the free energy of ion hydration still being relatively large.

In the following, we include the water solvent in the ten-

tative selection of reaction pathways from reactant 1 to the

hydride product 5.

Table 2 Summary of CP-MD simulations results

Models Summary of results Figures

REP pathway (Fig. 2a)

M1 and M2 Thermal fluctuation of species 2, low water density around H2, Ru-bonded molecule Fig. 5

M3 Rapid formation of species 5, water molecules expelled from Ru site, Ni–l-S2–Ru cleft closed Fig. 6

M4 Larger fluctuations of H–H distance in 2, but Ru-bonded H2 cleavage prevented by low solvation Fig. 7

M5 Formation of 4 stabilized by strong interactions with water Fig. 8

Ru pathway (Fig. 2b)

M6 Thermal fluctuation of species 1, high water density around Ru site Fig. 9

M7 Rapid formation of 4 stabilized by strong interactions with water, back to model M5 (4) Figs. 10, 11

M7a Thermal fluctuation of species 4 stabilized by strong interactions with water, back to model M5 (4) Fig. 12 (left portion)

M7b Conversion of 4 into 5, release of water molecules at lower density, back to model M3 (5) Fig. 12 (right portion)

Ni pathway (Fig. 2c)

M8 H2 dissociation from Ni site, back to model M6 (1) Fig. 13

M9 H2 cleavage produces species 4, back to model M5 (4) Fig. 13

M10 Slow recovery of H2 associated with Ni species 8 constrained by high spin Fig. 13

See Fig. 2 for the identification of the species

154 J Biol Inorg Chem (2012) 17:149–164

123

REP mechanism

In the following models, the Ru-bonded water molecule in

1 is replaced by a H2 molecule (species 2), which is acti-

vated by the interactions with Ru and possibly by long-

range interactions with Ni.

Simulations M1 and M2

The REP mechanism was investigated, starting from the

simulation of compound 2 (Fig. 2a), i.e., the (g-H2)–Ru

adduct, in the presence of two NO3- anions and several

water molecules mimicking the water solvent (simulation

M1) starting from the configuration displayed in Fig. 3 (see

‘‘Methods’’ and Table 1 for details of the model

construction).

The simulation of this system at room temperature

(T = 300 K) and water density imposed by the stage of

constant volume simulation (simulation M1, see Table 1)

shows that the H2 adduct to Ru (2) is stable up to room

temperature with the geometry summarized in Fig. 5c.

In Fig. 5a, the evolution with time of several distances is

displayed. At low temperature (T \ 300 K) the H2 mole-

cule displays a H–H distance of 0.94 ± 0.03 A, larger than

the equilibrium distance of the isolated H2 molecule

(0.75 A) and consistent with the distance in the relaxed

structure (0.96 A). This latter value is slightly larger than

that observed for the H2-adduct intermediate identified for

Ni(xbsms)Ru(CO)2Cl2 [H,H-xbsms is 1,2-bis(4-mercapto-

3,3-dimethyl-2-thiabutyl)benzene] [7]. In this latter case

the H–H and H–Ru distances are 1.84 and 1.76 A,

respectively, when the formal oxidation state of Ni is I. In

our calculation, with oxidation state II for both metal ions,

the distances are 1.96 and 1.69 A, respectively, indicating a

larger antibonding character for the H–H bond when the

oxidation state of the complex is higher. Indeed, the ligands

in the Ni(xbsms)Ru(CO)2Cl2 complex favor H2 evolution

rather H2 cleavage.

At T = 300 K the H–H distance transiently displays

values about twice the isolated equilibrium distance

(1.3 A), thus approaching a bond cleavage. The evolution

with time and with temperature of the H–M distances, with

H belonging to the Ru-bonded H2 molecule and M indi-

cating either Ni or Ru atoms, shows that the H2 molecule is

g-bonded to Ru, with the H–H bond rotating approximately

around the axis going from the H–H bond center to Ru (see

the in vacuo relaxed configuration in Fig. 5c). This rotation

starts occurring at T [ 150 K, but even at room tempera-

ture is hindered by a barrier encountered when the H–H

segment tends to be parallel to the Ru–Ni segment. This

behavior represents the thermal libration of the H–H bond

around the (g-H2)–Ru axis, with significant elongation of

the H–H bond.

The H2 molecule is polarized by the interactions of H

atoms with Ni, these latter interactions being modulated by

the H2 molecule libration described above. This effect can

be measured by computing the electron charge within the

atomic basins spanned by the two atoms of the H2 molecule

Fig. 4 Species 1, 2, and 5 (the

latter in two forms) from

Fig. 2a. Energies are in

kilojoules per mole and 1 is the

zero reference value. Energy

values are in vacuo (first value),

including dispersive interactions

(second value), and accounting

for the proton hydration

experimental free energy

(third value)

J Biol Inorg Chem (2012) 17:149–164 155

123

in different orientations (see ‘‘Methods’’ for details). When

the molecule is in the local minimum (the H2 molecule is

almost perpendicular to the Ni–Ru segment, like in the in

vacuo relaxed configuration, Fig. 5c) the two atoms have

the same charge of 1.0e, whereas in the case of an almost

parallel H2 molecule, for instance, when the H1–Ni dis-

tance is 2.5 A and the H2–Ru distance is 1.6 A, the charges

are 0.8e and 1.2e for H1 and H2, respectively. This shows

that the H2 molecule is polarized when it is partially ori-

ented along the Ru–Ni axis. However, the H atom closer to

Ni, thus geometrically closer to the final H-bridging

hydride, is less negatively charged than that closer to Ru.

A graphical inspection of the configurations spanned at

T = 300 K by simulation M2 (performed at a pressure of

10 bar) showed that even at such high pressure a signifi-

cantly large cavity is present in the water solvent on both

sides of compound 2. This effect can be measured by the

RDF of atoms pairs, where the two atoms in each pair

belong, respectively, to the solute and to water molecules

(either H or O atoms). Noticeably, the time evolution of

monitored distances at T = 300 K is very similar in sim-

ulations M1 and M2, despite the large difference in the

volume of the sample (see Table 1). In Fig. 5d, the RDF is

displayed for simulation M2 and for different solute–sol-

vent pairs. The extent of the empty space around the solute

center is measured by the RDF of pairs involving atoms

bonded to metal ions (Nam, S, and the C atoms of the

benzene ring, Car), and any atom in the water molecules.

This latter RDF (purple curve) shows that the bulk water

lies beyond 4.5 A and that within this distance no structure

for water is present (no significant peaks in the RDF). The

Ru-bonded H atoms show a different RDF involving O

atoms of water molecules in the solvent (the green line):

the peak at 2.5 A shows the presence of persistent water

molecules around the H2 molecule (thus representing

configurations like 3 in Fig. 2a), but at a larger distance the

density of pairs is lower than that observed by the other

metal-bonded atoms (the purple line). This is particularly

unexpected, with, in theory, the Ru-bonded H2 molecule

being largely exposed to the solvent (see Fig. 3). Com-

paring the RDF for H–O pairs in the water solvent (the red

line) with the H(H2)–O pairs (green line), one can see that

the first peak of the former RDF is at a distance of about

1.5 A, a distance less than that of the unique peak associ-

ated with the latter RDF. This shows that a distance

allowing a significant proton exchange, as occurs in bulk

Fig. 5 Results for models M1 and M2 (species 2 in water at low and

high density, respectively). a Time evolution of several interatomic

distances in simulation M1. b Time evolution of total electronic

energy, with the minimal energy sampled (Emin) as the reference

energy; the horizontal bars represent the thermal energy

E = Emin ? (3Na - 6)RT associated with each simulated tempera-

ture (50, 150, and 300 K, also indicated in b in the given time ranges,

see Table 1), with Na the number of atoms in the simulated sample.

c Structure of the in vacuo relaxed initial configuration (2 in Fig. 4).

d Radial distribution function (RDF) for compressed model M2 and

for several atoms pairs, computed at T = 300 K (see the text for

details). Pairs H(H2O)–O(H2O) red (intramolecular pair excluded),

H(H2O)–O(NO3-) blue, H(H2)–O(H2O) green, and Nam/S/Car–H/

O(H2O) purple

156 J Biol Inorg Chem (2012) 17:149–164

123

water (1.5 A), is never reached by the H atoms of the Ru-

bonded H2 molecule, even at high water density (model

M2).

As for the two NO3- anions, they display a first-shell

water layer similar to bulk water (compare the blue and the

red lines in Fig. 5d), thus showing that nitrate anions are

significantly taken apart from the positively charged com-

plex by the few (67) water molecules included in the

model. Nevertheless, nitrate anions are kept close to the

axis where they were located at the beginning of the sim-

ulation (see Fig. 3), that is, the axis crossing the Ni atom

and approximately perpendicular to the plane formed by

the Ni ligand atoms. This persistence indicates that the

electric field associated with the cation is higher on the Ni

side than on the Ru side of complex 2.

Summarizing, simulations M1 and M2 confirm the sta-

bility of species 2, where the Ru-bonded water molecule of

1 is replaced by H2, as was indicated by in vacuo relaxa-

tion. The analysis of the behavior of the cation environ-

ment suggests the presence of a cavity in the solvent

around species 2, with three consequences: a few water

molecules approach the Ru-bonded H2 molecule, but at an

O–H average distance larger than that sampled in the bulk

solvent; the cavity around the Ru-bonded H2 molecule is

large enough to seize other H2 molecules of the reactant;

the NO3- anions approach the Ni side of the bimetallic

complex.

Simulation M3

A model in which one of the H atoms of the Ru-bonded H2

molecule is moved towards the closest water molecule,

mimicking configuration 4 displayed in Fig. 2a, was built

and simulated (model M3, hereafter).

Simulation M3 shows the rapid formation, already at

T = 100 K, of the bridging hydride species 5 (Fig. 2a and

product in Eq. 2), summarized in the in vacuo relaxed

configuration displayed in Fig. 6c. The final geometry is

similar to that reported by the neutron diffraction experi-

ment on species 5: the H–Ru and H–Ni distances at

T = 300 K are 1.66 ± 0.03 and 1.95 ± 0.1 A, respec-

tively, compared with the experimental values of 1.68 and

1.86 A. The time evolution of the M–H distances (Fig. 6a)

clearly depicts the transformation of species 4 into 5, thus

showing that 4 in the water solvent at the simulated density

lies on the product side along the reaction coordinate of the

REP mechanism.

The movement of the H atom initially bonded to Ru

towards Ni is accompanied by a wide rearrangement of the

L and L0 ligands. In particular, the methylbenzene ring

Fig. 6 Results for model M3 (initially species 4 in water at low

density). a Time evolution of several interatomic distances. b Time

evolution of total electronic energy (horizontal bars are the thermal

energy at the indicated temperatures, T = 50, 100, 200, and 300 K,

respectively). c Structure of the in vacuo relaxed final configuration.

d Radial distribution function (RDF). Pairs H(H2O)–O(H2O) red(intramolecular pair excluded), H(H2O)–O(NO3

-) blue, H(Ni–H–

Ru)–O(H2O) green, Nam/S/Car–H/O(H2O) purple

J Biol Inorg Chem (2012) 17:149–164 157

123

rotates around the axis approximately perpendicular to the

plane formed by Ni, Ru, and the bridging H, a movement

displayed also in the in vacuo relaxation (compare Figs. 5c,

6c). This rotation closes the cleft around the H bridging

atom, expelling the water solvent from the coordination

side occupied by the bridging H atom (see Fig. 6d and

discussion below). The Ni coordination is square-pyrami-

dal, with the H atom at the axial position. The Ru coor-

dination is nearly octahedral, with the g6-C6Me6 ligand

mimicking three bonds. Remarkably, this final state is

obtained with no spin polarization (Sz = 0). The replace-

ment of the Ru-bonded water molecule by H2 and the

consequent desolvation of that side of the cationic complex

(as measured by the RDF displayed in Fig. 6d) make

possible the movement of the benzene ring. The RDF of

the bridging H atom in the product is lower than that of the

H2 adduct (compare Figs. 5d, 6d). This is accompanied by

the movement of the nitrate anions, which are fully sol-

vated, far from the cation (the location of maxima in the

RDF of nitrate O atoms approaches that of bulk water,

compare the blue and red lines in Fig. 6d). The release of

counterions is expected as an effect of the decrease in net

positive charge of the cation. Nevertheless, since nitrate

anions always interact with water molecules, in this case

the movement of nitrate anions is accompanied by the

movement of water molecules away from the cation.

Since models M1 and M3 have the same number of

atoms and the same supercell size, the comparison between

the T = 300 K average total energies (Figs. 5b, 6b) pro-

vides an indication of the energy change in the reaction

2 ? H2O ? 5 ? H3O? at T = 300 K. In water solution

and in the presence of the counterions, this energy change

is still negative (about -300 kJ/mol), but is less negative

than the value estimated at T = 0 K (at least -350 kJ/mol,

Fig. 4), where the change in solvation for the two cations, 2

and 5, respectively, was not taken into account.

The low-temperature stage of simulation M3 allowed us

to estimate the minimal energy of 4 before its transfor-

mation into 5. The difference between the minimal ener-

gies of simulation M3 (i.e., the reference energy in Fig. 6b,

species 4) and that of simulation M1 (the reference energy

in Fig. 5, species 2) results in an estimate of 120 kJ/mol for

the transformation, at T = 0 K and at the given water

density, of 2 into the metastable species 4.

Simulation M4

In simulation M4, the cavity in the solvent exploited in

simulations M1 and M2 (species 2) was filled with three

additional H2 molecules, mimicking a possible excess of

reactant and avoiding possible spurious effects of the

vacuum on both solvent and solute. Moreover, the simu-

lation was performed in the Sz = 1 configuration.

None of the added H2 molecules were found bonded to

any of the other atoms in the system at T = 300 K. The

comparison between the RDF for pairs involving any atom

in the complex cation and any H in the three additional H2

molecules with the RDF involving the same solute atoms

and water H atoms (data not shown) shows that the added

H2 molecules are kept closer to the complex cation than the

water solvent.

Because of the Sz = 1 spin configuration used in simu-

lation M4, the H–H bond distortions are larger than in

simulations M1 and M2. Indeed, configurations with a H–H

distance of 1.6 A, which is about twice the equilibrium

H–H distance observed in simulations M1 and M2, were

sampled, but, like in models M1 and M2, the H–H bond

cleavage is never achieved and these distorted structures go

back to the reactant state represented in simulations M1

and M2. This is also confirmed by the average H–H dis-

tance at T = 300 K: 1.1 ± 0.2 A. Nevertheless, the anal-

ysis of distorted configurations allows one to understand

environmental effects eventually assisting the H–H bond

heterolysis.

The structure in Fig. 7 displays such a large H–H bond

distance, and water molecules with distances within 2.5 A

are also displayed. Also the NO3- anions are represented

together with a water molecule that is docked, for the whole

duration of the simulation at T = 300 K, between one

NO3- anion and the H2 molecule bonded to Ru. Despite the

presence of several basic groups at short distance from the

H atoms involved in the distorted H–H bond (two water

molecules and one of the S atoms of the ligand), the H–H

bond appears homolytically, rather than heterolytically,

cleaved: both H atoms interact with basic groups, mainly

because they are still interacting with the same Ru atom (the

Fig. 7 Configuration displaying large extension of the H–H bond

(1.5 A) for the Ru-bonded H2 molecule in species 2 in simulation M4

(high water density and Sz = 1)

158 J Biol Inorg Chem (2012) 17:149–164

123

H–Ru distances in the displayed structure are both 1.6 A).

Moreover, statistically the approach of water O towards the

H atoms is very similar to that observed in simulation M2 at

the same temperature: the peak in the H(H2)–O(H2O) RDF

(data not shown) is at a closer distance than in simulation

M2 (about 2 instead of 2.5 A), but this distance is still larger

than that of the bulk water (about 1.5 A).

The persistence of a water molecule between NO3- and

Ni on the same side of the H2–Ru bonded molecule ham-

pers the possible axial approach of the NO3- anion towards

Ni and the consequent stabilization of an octahedral Ni

coordination at high spin. This approach occurs on the

opposite side, where one of the NO3- O atoms is bonded to

Ni (see Fig. 7) displaying an average O(NO3-)–Ni distance

of 2.14 ± 0.09 A at T = 300 K. The full high-spin octa-

hedral coordination of Ni is, therefore, prevented by the

hindrance around Ni on the side where the closest approach

of H2 occurs, together with the stabilization of the square-

planar Ni coordination on the opposite side.

Simulation M5

Instead of manually moving one of the H atoms away from

the Ru-bonded H2 molecule towards the closest water

molecule (model M3), we used a different, less biased,

approach to trigger the H2 cleavage (see ‘‘Methods’’ for

details). The low-temperature simulation of species 2, i.e.,

the T = 50 K trajectory of model M3, with the addition of

four H2 reactant molecules, was followed by a two-electron

oxidation and the trajectory of atoms, after relaxation, was

followed in the oxidized state up to a configuration where

the H–H bond of the Ru-bonded H2 molecule was cleaved.

Once this state is reached, the system is reduced to its

original charge state, and the simulation was started again

after the initial forces had been set to zero.

As revealed by maps of the electron density difference

between reduced and oxidized states (data not shown),

upon oxidation the two electrons are removed from the rbond of the Ru-bonded H2 molecule. After a few femto-

seconds of the trajectory at T = 50 K in the oxidized state,

one of the H atoms (H1) of the Ru-bonded H2 molecule is

captured by a water molecule close to the Ru center. The

other H atom (H2) remains bonded to Ru. In Fig. 8a, the

comparison between the time evolution of selected dis-

tances before and after the oxidation shows that the H2

cleavage is achieved with the transient oxidation, and that

the original H2 molecule is not recovered once the system

is reduced. The large distance between the Ru-bonded H

atom (H1 in the figure) and Ni shows that the system easily

achieves the state corresponding to species 4, whereas the

formation of the product (5) is not achieved because of the

strong interaction between the H2 atom (now belonging to

the closest water molecule) and H1. This shows that the

single hydride H1 atom (bonded to Ru) displays competing

interactions between protons (this latter associated with

water molecules) and Ni. Only when hydride–proton

interactions are released (like in the low water density

model M3) is Ni allowed to come into the interaction

sphere of the hydride H and the complete formation of 5 is

allowed. Nevertheless, the back-formation of 2 is never

observed, thus showing, like in the M3 case, that 4 is on the

product side of the reaction in Eq. 2. The nature of 4 as

obtained in model M5 is very similar to that of the terminal

Ru hydride species obtained in DFT calculations of similar

NiRu bimetallic complexes [7, 18]. The average Ru–H

distance obtained at T = 300 K by model M5 is

1.63 ± 0.03 A, to be compared with 1.60 A in the geom-

etry optimized structure of Ni(xbsms)Ru(CO)2ClH, which

is in the same oxidation state of model M5.

The energy change at T = 50 K in the M5 simulation

(Fig. 8b) indicates a positive value of 400 kJ/mol for the

minimal energy change in the transformation of 2 into 4.

Comparing this value with that obtained at the lower water

density (120 kJ/mol, see the end of ‘‘Simulation M3’’), one

can see that the water density has a large influence by

increasing the energy gap to reach the metastable species 4.

The Ru pathway

The results of simulations M1–M5, where a H2 molecule

was added to Ru, displacing the Ru-bonded water molecule

Fig. 8 Results for model M5 (initially species 2 in water at high

density). a Time evolution of several interatomic distances. b Time

evolution of total electronic energy

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123

observed in the characterization of complex 1, indicated

that the Ru-bonded H2 molecule, although stably interact-

ing with Ru in complex 2, is not prone to a heterolytic

cleavage and possible basic groups nearby rarely approach

the H2 molecule to efficiently assist the cleavage.

The presence of the Ru-bonded water molecule, while

hindering the approach of H2 molecules towards Ru, is

expected to provide an in-place basic group. For the fol-

lowing simulations, the Ru-bonded water molecule was

initially in the position revealed by the experiments

(species 1 in Fig. 2).

Simulation M6

The Ru-bonded water molecule remained bonded to Ru up

to T = 300 K (red line in Fig. 9a) and an extended network

of H bonds centered around the Ru-bonded water molecule

was observed for the whole T = 300 K trajectory (Fig. 9c).

The measure of this network is given by the RDF dis-

played in Fig. 9d. In this plot the RDF of pairs involving

the Ru-bonded H2 molecule and water O atoms in Fig. 9d

(the green line) is replaced with that involving H(H2O–

Ru)–O(bulk-H2O). It can be noticed that the first peak in

the RDF shown by the green line is at shorter distance

compared with the same RDF computed within the bulk

water molecules (the red line), thus indicating that the

Ru-bonded water molecule is more acidic towards bulk

water than bulk water itself. Indeed, one of the two H

atoms of the Ru-bonded water molecule (H2) is transiently

moved towards the closest water molecule (Oc1, blue line

in Fig. 9a), this latter molecule acting as a base. The

Ru-bonded water molecule is asymmetric because of the

different types of interactions with Ni (by H1) on one side

and with nearby water on the opposite side (H2): the

H1–Ow distance at T = 300 K is 0.99 ± 0.01 A, and is

thus only slightly shorter than that of a persistent O–H

bond in bulk water molecule (1.00 ± 0.02 A). On the other

hand, the H2–Ow distance is 1.12 ± 0.08 A, with H2

jumping within two positions (light-blue line in Fig. 9a), as

is the case for the H atoms involved in H-bond exchange

within the bulk water molecules.

This time evolution shows a partial dissociation of the

Ru-bonded water molecule (light-blue line in Fig. 9a) and

the partial proton donation is concerted with the partial

acquisition of a H atom from a third water molecule nearby

(Hc2), this latter opposing Ni with respect to Ow. The main

difference between the activated Ru-bonded water mole-

cule and the Ru-activated H2 molecule of models M1–M5

Fig. 9 Results for model M6 (species 1 in water at high density).

a Time evolution of several interatomic distances. b Time evolution

of total electronic energy. c Final structure at T = 300 K. d Radial

distribution function (RDF). Pairs H(H2O)–O(H2O) red (intramolec-

ular pair excluded), H(H2O)–O(NO3-) blue, H(H2O–Ru)–O(H2O)

green, Nam/S/Car–H/O(H2O) purple

160 J Biol Inorg Chem (2012) 17:149–164

123

is that the former activation is assisted by water molecules

nearby and is largely affected by water structure, whereas

in the latter case the water molecules are farther in space

and are, therefore, less affected by H2 activation.

Simulation M7

Simulation M6 addressed the presence of basic water

molecules near Ru, transiently produced by the Ru-bonded

water molecule. To investigate if this basic group can assist

the proton extraction from a heterolytically cleft H2 mol-

ecule, one further H2 molecule was added to the final

configuration obtained with simulation M6. The closest

H–Ru distance was set to 2.3 A and the approach by H2 to

Ru was performed from a direction almost perpendicular to

the plane formed by Ni, Ru, and the Ru-bonded water

molecule.

Several simulations of this system showed that the H2

molecule, initially in contact with Ru, moves far from Ru,

showing no propensity for Ru association or for interac-

tions with any atom in the complex.

Therefore, in order to initially stabilize a close-contact

H2–1 complex, mimicking species 6 in Fig. 2b, the system

was oxidized (charge ?2) and, once the forces were suf-

ficiently small in the oxidized state, the system was

reduced (charge 0), and the simulation was started in the

reduced ground state (simulation M7, hereafter).

The oxidation of the complex favors a rapid cleavage of

the H2 molecule in contact with Ru. This occurs during the

first 0.1 ps of the simulation at T = 50 K.

The trajectory in the reduced state (Fig. 10a) shows that

once the H2 molecule is cleaved, one of the H atoms forms

a stable bond with Ru (the green line), whereas the other H

atom is captured by a water molecule, indeed the same

basic water molecule that in model M6 attracted one of the

H atoms belonging to the Ru-bonded water molecule. The

water molecule that extracted H2 remains close to the

hydride H1 atom (purple line), but the H2–H1 distance

never goes below 1.2 A (like in model M5), thus showing

that the product of the H–H cleavage cannot form a H2

molecule again. This shows that, like in model M5, the

process produced species 4. The initially Ru-bonded water

molecule dissociates from Ru and moves towards the bulk

(light-blue line). Another water molecule strongly interacts

with the H1 hydride atom. The latter residual interaction

between the hydride H1 atom and water molecules pre-

vents the formation of the H1–Ni bond, the formation of 5,

and the desolvation of the lower-charge cation (see

‘‘Simulation M3’’ and below).

In contrast with all the previous simulations, the energy

change (Fig. 10b) at low temperature (T = 50 K) between

the initial state before oxidation (1 weakly interacting with

H2) and the initial state after oxidation (4 strongly

interacting with nearby water molecules) is close to zero.

Comparing the energy of model M7 at T = 300 K (end of

plot in Fig. 10b) with that of model M1 (end of plot in

Fig. 5b), one can see that the thermal energy changes

(about 1,200 kJ/mol) in the two cases are similar, with

larger fluctuations in the former case, as expected for a

metastable state.

In Fig. 11, the initial (Fig. 11a) and final (Fig. 11b)

configurations in the reduced state are displayed. It can be

noticed that the entire thermalization process allows the

relaxation of an unstable hydride species towards a stable

one, with residual H–H interactions involving the hydride

H atom and protons belonging to water molecules.

Simulation M3 suggested that the production of the final

bridging hydride species 5 is favored by the low density of

water molecules around the cationic complex. This is

confirmed by models M7a and M7b, where the water

density was progressively decreased from the value of

simulation M7 to that of simulation M3 (see Table 1).

The time evolution of distances (Fig. 12a) shows that

only the lowest water density (the same density as for

simulation M3, the right-hand portion of the plot) allows

the complete formation, at T = 300 K, of species 5,

whereas at the higher density the residual interactions of

the hydride H atom with protons of water molecules nearby

prevent the interaction with Ni. This effect, similar to what

is observed in simulation M5, shows that Ni competes with

water H atoms in the interaction with the hydride H atom.

Fig. 10 Results for model M7 (H2 adduct of species 1 in water at

high density, mimicking species 6 in Fig. 2b). a Time evolution of

several interatomic distances. b Time evolution of total electronic

energy

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123

The time evolution of the total electronic energy

(Fig. 12b) shows that the energy change at T = 300 K in

the transformation of species 4 into species 5 (the change in

average energy in the second half of the T = 300 K tra-

jectory) is about -200 kJ/mol, whereas the energy change

due to the density change of species 4 (the difference

between the T = 300 K average energies of species 4 at

the two values of water density) is about 500 kJ/mol. This

indicates that once the environmental conditions of the

cation are changed, at the expense of a large amount of

external work, the chemical transformation of the cation

spontaneously occurs at room temperature and in less than

1 ps.

The Ni pathway

Similarly to the models for the Ru pathway, initial models

for the Ni pathway, resembling species 8 in Fig. 2c, were

built. The Ru-bonded water molecule identified by

experiments was kept in the model, and one of the four H2

molecules inserted in model M4 was moved close to the

axial position that is left open around Ni by simulation M4.

The closest H–Ni distance was 1.97 A in the initial

configuration.

Simulation M8

The simulation of species 8 in the Sz = 0 spin configu-

ration provides a rapid dissociation of the initially close

H2 molecule from Ni (blue line in Fig. 13a on the left),

keeping the H2 molecule intact (red line). The simulation

in the Sz = 1 spin configuration displays similar behavior

and a larger energy (data not shown). These simulations

show that there are no energy barriers in the pathway

from species 8 to species 1, whatever the spin configu-

ration, similarly to what is shown by simulation M5 for

the Ru site. The approach of H2 to both the metal centers

is probably controlled by diffusion. Nevertheless, in most

of the simulations reported here, the approach of H2

towards the axial coordination position of Ni was partially

hindered by the persistence of the nitrate anions, these

latter anions competing for the same sites. However, this

latter result may be an effect of the small size of the

simulated sample.

Fig. 11 Results for model M7. a Initial structure after oxidation.

b Final structure of 4 in the reduced state

Fig. 12 Results for models M7a and M7b (continuation of model M7

at lower water density). Model M7a is on the left and model M7b is

on the right. a Time evolution of several interatomic distances.

b Time evolution of total electronic energy; the reference energy is

the same as in Fig. 11b (model M7)

162 J Biol Inorg Chem (2012) 17:149–164

123

Simulation M9

As in the Ru pathway, the H–H bond in the H2 molecule in

contact with Ni was cleaved by oxidizing the system

(imposing a supercell charge of ?2).

Upon oxidation, the H1–H2 distance rapidly reaches the

value of 1.2 A that was found in previous models, a value

beyond which there is no back-formation of the original H2

molecule. The H1 atom during the oxidized trajectory

rapidly moves from the Ni to the Ru site, forming species 4

(as displayed by the green line in Fig. 13a). Noticeably,

this occurs with an energy decrease in the reduced state

(Fig. 13b) at both low (T = 50 K) and high (T = 300 K)

temperatures, showing that species 4 is more stable than 8

in the low-spin configuration. Finally, the Ru-bonded water

molecule, which extracted the H2 atom from the initially

Ni-bonded H2 molecule (see also ‘‘Simulation M10’’), is

ejected from its original coordination site (purple line). The

hydride H1 atom does not become close to Ni because of

residual interactions with water molecules, similarly to

what was observed in simulation M7. This simulation,

therefore, converges to the end of simulation M7.

Simulation M10

Another simulation of species 8 was performed by inter-

rupting the oxidized trajectory before the exchange of the

hydride H1 atom between Ni and Ru and keeping the

system in a high-spin configuration (Sz = 1). As displayed

in the rightmost portion of the plot in Fig. 13a, the H1–Ni

bond distance (blue line) is kept, up to room temperature,

but the H2 atom initially moves towards the Ru-bonded

water molecule. The proton acquisition by the Ru-bonded

water molecule, which keeps its bond with Ru, is com-

pensated by the proton donation towards a nearby water

molecule (in a position similar to the basic water molecule

addressed in simulations M6 and M7). This proton

exchange maintains a high-spin Ni hydride form (9 in

Fig. 2c). Nevertheless, the time evolution of the H1–H2

distance denotes a smaller average value compared with

simulation M9 (the central plot in Fig. 13), indicating a

slow back-formation of the H2 molecule. A complete for-

mation of the original H2 molecule is prevented by the

high-spin configuration, which indeed has a high energy,

compared with the M8 simulation, even at low tempera-

ture. Therefore, when the low-spin configuration is

restored, the system rapidly forms the original H2 molecule

and behaves like simulation M8, with the H2 dissociation

from Ni and with the release of the interaction with the

Ru-bonded water molecule.

Simulations M9 and M10 show that species 9 and 8 lie

either on the side of reactant 1 or on the side of product 4.

In this latter case, the system goes towards the completion

of the Ru pathway.

Conclusion

A series of first-principles molecular dynamics simulations

in the Car–Parrinello scheme were performed to discrimi-

nate among different proposed pathways for the heterolytic

cleavage of the H2 molecule catalyzed by a simple water

soluble model compound mimicking the active site of

[NiFe] hydrogenase.

The calculations, summarized in Table 2, show that the

displacement of the Ru-bonded water molecule from the

reactant by the incoming H2 molecule forms an g-H2

adduct to Ru that weakly interacts with basic groups

nearby, both in the ligand (the S atoms) and in the water

surrounding the complex. This observation excludes the

mechanism for H2 cleavage following water replacement

by H2 in the Ru coordination sphere.

Calculations that keep the Ru-bonded water molecule

originally present in the crystal structure of reactant 1 show

that an extended network of H bonds is present, that the

water molecule bonded to Ru is prone to donate a proton to

a basic water molecule in the second coordination shell of

Ru, and that the H2 heterolytic cleavage can proceed via an

almost zero-energy process up to the H–Ru hydride species

4. These observations support a mechanism where the

second layer of water molecules plays an active role in the

H2 cleavage.

Fig. 13 Results for models M8 and M9 (initial H2 adduct to Ni,

species 8, in water). a Time evolution of several interatomic

distances. b Time evolution of total electronic energy

J Biol Inorg Chem (2012) 17:149–164 163

123

Similar exploitations of the pathway of H2 molecules

towards Ni show that in this case the H2 cleavage has larger

chance of going back to the reactant molecules. In all cases

where this latter back-reaction does not occur, the same

intermediate 4 of the Ru pathway is produced.

These calculations, therefore, show that the role of Ni in

the cation is in orienting the Ru-bonded water molecule,

favoring the presence of a basic water molecule on the side

of Ru assisting the H–H cleavage. When the heterolytic

cleavage is completed, the H–Ru species produced is

strongly stabilized by the release of water and counterions

from the cation. Under these latter conditions, the bridging

hydride product is formed and can be easily crystallized

from water solution.

By using the first-principles molecular dynamics tech-

nique, we investigated in detail this peculiar role of the

metal-bonded water molecule, and of the water molecules

in its close environment, as an essential means for proton

extraction away from the metal-activated H2 molecule. The

investigation of this simple biomimetic system in water

solution will serve as a basis for further studies of electron

transfer processes coupled with proton exchange with the

environment: this coupling is essential in biological and

biomimetic energy-harvesting devices inspired by

hydrogenase enzymes.

Acknowledgments This work was done within the project Eit040

of the Julich node of the Gauss Centre for Supercomputing (Ger-

many). A range of 32–128 parallel tasks were used on the Juropa

architecture. Partial support from task 21 (biohydrogen) of the

International Energy Agency Hydrogen Implementing Agreement is

acknowledged.

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