New insights into the reaction capabilities of His195 adjacent to the active site of nitrogenase

    New insights into the reaction capabilities of His195 adjacent to the active site of nitrogenase Ian Dance PII: DOI: Reference:

S0162-0134(16)30259-8 doi:10.1016/j.jinorgbio.2017.01.005 JIB 10147

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

26 September 2016 1 December 2016 3 January 2017

Please cite this article as: Ian Dance, New insights into the reaction capabilities of His195 adjacent to the active site of nitrogenase, Journal of Inorganic Biochemistry (2017), doi:10.1016/j.jinorgbio.2017.01.005

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New insights into the reaction capabilities of His195 adjacent to the active site of nitrogenase Ian Dance School of Chemistry, UNSW Australia, 2052

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New enhanced density functional calculations reveal that His195, strongly implicated in the mechanism of the enzyme nitrogenase, can undergo reversible proton transfers, dihydrogen bond formation, and H2 formation in its interactions with the active site FeMo-co. Keywords: Nitrogenase, mechanism, Density Functional calculations, hydrogen bond, dihydrogen bond, FeMo-co

Abstract The active site of the enzyme nitrogenase is FeMo-co, a C-centred Fe7MoS9 cluster, connected to the surrounding MoFe protein via ligands Cys275 and His442. Density functional calculations, involving 14 Dance_His195_reactions: JINORGBIO_2016_4 revised

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additional surrounding amino acids, focus on His195 because its mutation causes important reactivity changes, including almost complete loss of ability to reduce N2 to NH3. The N side-chain of His195 is capable of hydrogen bonding to S2B, bridging Fe2 and Fe6 of FeMo-co, believed to be the main reaction domain of nitrogenase. Details are presented for the possible ways in which protonated or deprotonated N of His195 interact with S2B or S2B-H or Fe2 or Fe2-H or Fe-(H2). Movements of the His195 sidechain allow formation of a significant short dihydrogen bond between N of His195 and H on Fe2: NH••H-Fe2, with H--H = 1.39Å. It is shown that a 180° rotation of the imidazole ring of His195 is not able to facilitate transfer of protons from the protein surface to FeMo-co. His195 is able to move H atoms to and from S2B, and the characteristics of H transfer between S2B and N of His195 are described, together with their dependence on the protonation state of His195 and the redox state of FeMo-co. The water molecule on the posterior N side of His195 can mediate proton transfer to and from the side-chain of Tyr228. The accumulated results suggest that protonated His195 could be the agent for the first, most difficult, transfer of H to bound substrate N2.

1. Introduction

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The enzyme nitrogenase converts N2 to NH3.[1, 2, 3, 4, 5, 6, 7, 8, 9] The catalytically active site where N2 binds and is hydrogenated is the FeMo-cofactor (FeMo-co), illustrated in Fig 1. Located in the MoFe protein of the dual protein enzyme, FeMo-co is a C-centred Fe7MoS9 cluster,[10, 11] with bidentate homocitrate chelated at Mo, His442 coordinated via imidazole at Mo, and Cys275 coordinated via cysteinate S at Fe1.[4, 12, 13, 14] Residue numbering through this paper is that of Azotobacter vinelandii protein, with atom labelling as in PDB 3U7Q.

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Fig 1. (a) FeMo-co, linked to protein via 275Cys at Fe1 and 442His at Mo. Atom labels are those of the Azotobacter vinelandii protein, PDB 3U7Q. (b) The partly helical chain containing His195 within hydrogen bonding distance of S2B. Carbon atoms of bidentate homocitrate are dark green; water oxygen atoms are orange.

Site-directed mutagenetic investigations have revealed the catalytic importance of a number of residues close to FeMo-co. One is -70, valine in wild-type protein. Changes in the shape and volume of this Val side-chain (to Ala, Ile, Gly) cause complementary changes in the reactivities of alternative substrates according to their size, indicating that these substrates bind and react at the section of FeMo-co covered by -70, that is the Fe2, Fe6 domain.[6, 15, 16, 17] Another critical residue is -195, histidine in wild-type protein. As shown in Fig 1 (b) the imidazole sidechain of this residue protrudes from a partly helical chain to within hydrogen bonding distance of S2B that bridges Fe2 and Fe6. An adjacent protruding side-chain is -Gln191 that is hydrogen bonded to an uncoordinated carboxylate arm of homocitrate. Modification of His195 demonstrates its significance to the enzymatic mechanism. Replacement by Tyr, Gln, Asn, Thr, Gly or Leu extinguishes diazotrophic growth,[18, 19] and Gln195 or Asn195 mutation results in almost complete loss (<1%) of N2 reduction ability.[19, 20] However, N2 is able to inhibit the reduction Dance_His195_reactions: JINORGBIO_2016_4 revised

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of protons and of acetylene by the Gln195 and Asn195 proteins, [21] suggesting that N2 is able to coordinate to key binding sites of FeMo-co even though it cannot be hydrogenated to NH3. Additional biochemical and spectroscopic data for other substrates and for inhibitory interactions are available on proteins mutated at residue -195.[20, 22, 23, 24, 25, 26] The crystal structure of the Gln195 protein reveals that the glutamine side chain is able to hydrogen bond with S2B with geometrical similarity to the wild-type protein.[24] Prominent amongst the models suggested for the key role of His195 is proton transfer from NH of His195 to bound substrate, although it is also recognised that this path is unable to provide the multiple protons required for each catalytic cycle with the stoichiometry N2 + 8H+ + 8e-  2NH3 + H2.[27] Hydrogen bonding roles for -NH/-N of His195 are proposed for the structures of allyl alcohol / allyl amine trapped on FeMo-co and characterised spectroscopically when propargyl alcohol / amine are reduced with Ala70 modified enzyme.[28, 29, 30, 31, 32] These accumulated data and speculative interpretations enjoin an atom-level examination of the relationships between His195 and FeMo-co. In this paper I report density functional calculations of an extensive model (ca 260 atoms) containing FeMo-co and all relevant surrounding residues, including the possible protonated forms of His195 and its hydrogen bonding partners, and relevant hydrogenated forms of FeMo-co. The focus is on hydrogen bonding by His195 and simulation of possible proton/hydrogen transfer reactions via those hydrogen bonds. A previously unrecognised dihydrogen bond N-H••H-Fe is characterised, together with its ability to generate H2, possibly bound to Fe. I describe also the reversible reaction that transfers H between His195 and S2B of FeMo-co.

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Hoffman and Seefeldt[33, 34] have demonstrated experimentally the link between the binding of N2 and the release of one molecule of H2, originally proposed from the kinetic analysis by Thorneley and Lowe.[35] The systems described in this paper do not involve coordinated N2 or reacting N2, and the chemical mechanism for the linked binding of N2 and release of H2 is not addressed here. My objective here is to explore and define the reaction pathways that could be undertaken by His195 in the mechanisms of the many hydrogenation reactions known for nitrogenase.[1, 7, 36, 37] In the absence of N2 the enzyme reduces protons to form H2.

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The Bronsted convention formalises some of the processes considered here as proton transfers. The hydrogen chemistry of FeMo-co is best described in terms of movements of hydrogen atoms.[7, 38] In addition, variation of the electron population of FeMo-co (by electron transfer from the P-cluster) is a fundamental tenet for the mechanism of nitrogenase. For these reasons, calculated atom charges are reported here in order to clarify the extent and variation of bond polarisation during the processes described.

2. Computational procedures 2.1. Density functional methods Density functional (DF) calculations use the DMol methodology of Delley, [39, 40, 41, 42, 43] with accurate double numerical (dnp) basis sets.[42] These numerical basis sets avoid the need for basis set superposition corrections, and provide reliable energies at long intermolecular distances.[44] This advantage of the DMol methodology largely obviates the need for empirical treatment[45] of the longrange dispersion energies that are involved when the computational model is composed of separate molecular units. The calculations were all electron, spin-unrestricted, with no imposed symmetry, and employed the BLYP functional.[46, 47] The real-space cutoff for calculation of atomic basis sets was 4.76Å, and a fine integration mesh was used. The electronic state of FeMo-co was controlled through the input specification of the signs and magnitudes of the Fe spin densities to be used at the start of the SCF Dance_His195_reactions: JINORGBIO_2016_4 revised

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convergence calculation, and refined in the subsequent optimisation: this is equivalent to the commonly used broken-symmetry methodology. The stable electronic state adopted for FeMo-co is defined by the spin sign set {+, -, +, -, +, +, -} for Fe1 through Fe7.[48] The reported net spin S for each species was controlled through occupations of the unrestricted  and  orbitals in the usual way. The COSMO continuum solvation model[49, 50, 51] was applied, with a dielectric constant of 5. Zero point energies and thermal corrections were not calculated: potential energies are reported. Transition states were obtained by the pragmatic procedure previously described,[52, 53, 54] involving mapping of the potential energy surface. The transition states were confirmed by the criteria of zero gradient and demonstrated direct connection with the reactant and product on the potential energy surface. Atom partial charges were calculated using Mulliken population analysis[55] and the Hirshfeld partitioning scheme.[56]

2.2 Model development

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The validity of this DMol/BLYP methodology has been demonstrated through calculations of a variety of experimental data, including the structures of metal sulfide clusters,[57, 58] FeMo-co,[48, 59, 60] the properties of systems with H, H2 and/or N2 bound to Fe,[61, 62, 63] and for the hydrogen bonds in a variety of water aggregates.[54] Some of the hydrogen bonds and reaction steps that will be elaborated in this investigation are comparable with hydrogen bonds and proton transfer reactions involving coordinated H2 that have been observed and characterised thermochemically in other coordination and organometallic systems. Therefore relevant validations were made by calculating these additional experimental data using my computational methods, and the comparative results are summarised in the Supplementary Information. There is acceptable agreement, given that calculated potential energies are being compared with thermochemical data. Coordinates for the structures and transition states described in this paper are provided in the Supplementary Information.

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The section of the MoFe protein used in the calculations to follow is diagrammed in Fig 2, where the yellow background marks the region of most interest surrounding His195. Residues 65 to 71 which constitute the helix across the front face of FeMo-co are further from His195 but are included here because they are crucial for the subsequent modelling of steps at the front face. Residues Arg277, Ser278 and Tyr229 are linked with the FeMo-co ligand Cys275, and are included because they surround a putative substrate ingress route[3, 64, 65] near FeMo-co, also included in subsequent calculations. The net charge on the model of Fig 2 (a) is -1 when FeMo-co is at its resting redox level, core [Fe7MoCS9]-1: the net spin is S=3/2.

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(a) Model 1

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(b) HOH531 surroundings.

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Fig. 2. (a) Components of the MoFe protein included in the density functional model 1. Residues 65 to 71 (drawn separately, green background) are actually located in the helix that extends horizontally across the Fe2,Fe3,Fe6,Fe7 face of FeMo-co, and linked by two sets of hydrogen bonds (broken lines) through the side chain of Arg96 to S5A. The yellow background highlights His195 and its surrounds: the side chain of Phe381 is behind S2B. Residues are truncated where they extend away from the region of interest: C atoms are bold. (b) The four tetrahedral hydrogen bonding connections around HOH531, and four tetrahedral positions at OH of Tyr281. All amino acids are in the  chain of the MoFe protein.

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Water 531, located at the backside of His195 (distal from S2B) is significant. Analysis of the resting crystal structure (PDB 3U7Q) shows four feasible hydrogen bonding connections (Fig 2 (b)), tetrahedrally arrayed and therefore presenting various possible locations for its H atoms. There are also four tetrahedral positions around OH of Tyr281, allowing two possibilities for the H atom connection with HOH531.

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The model, 1, was extracted from PDB 3U7Q, hydrogenated and appropriately truncated, and then optimised. Initial optimisation cycles incorporated fixed positions for key C atoms and HOH531, which were subsequently released in final cycles. Small realistic movements occurred, generally as expansions relative to the centre of FeMo-co, and the model used as the starting point for the reactions to be investigated has C--Cc distances and Cc--HOH531 within 0.5Å of the crystal distances. For the simulations of reactions it was necessary to fix C of His442, and C the reaction domain were also fixed. Since His195 and its variability is the focus of this investigation the c--C), and remained within 0.2Å of the crystal position of its C position. The flexibility of the FeMo-co core occurred as some small extensions (up to 0.45Å) or contactions (up to 0.08Å) of the Cc-Fe2 and Cc-Fe6 bonds, caused by ligation of Fe2 and/or Fe6. Details are provided in the Supplementary Information S3. Also, an H atom bonded to S2B increases the S2BFe2 and S2B-Fe6 distances, as expected. This plasticity of FeMo-co is analogous to that calculated for other ligated forms of FeMo-co, and is consistent with experimental data.[66, 67, 68]

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3. Results

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3.1. Rotatory flexibility of the His195 side-chain

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Because the imidazole ring of His195 can form hydrogen bonds from its two N atoms with both S2B and HOH531, it might be thought[27] that a rotatory flip of this ring could be a mechanism for transferring protons between HOH531 and S2B. The relevant side-chain rotation that keeps the imidazole ring in the same vicinity is rotation about the C-C bond, and this has been investigated by density functional simulation within the full model 1. No serious geometric interference from other protein atoms occurs when His195 is rotated through 360° about C-C. Rotation by ca 90° in either direction places the face of the imidazole ring towards HOH531, with no opportunity for conventional hydrogen bonding, and with potential energy penalties of 17, 15 kcal mol-1. On energy minimisation there is reversion to the resting structure. Rotation by ca 180° directs C-H towards HOH531, thwarting hydrogen bonding, and directs C-H towards S2B, as shown in Scheme 1. This rotamer is at relative energy +17 kcal mol-1. It is evident that without more extensive movement of the His195 residue, involving movements of C and C and therefore of the helix (Fig 1 (b)), an imidazole flip to transfer protons from backside of His195 to FeMo-co is not feasible.

Scheme 1. The geometrical consequences of flipping the imidazole ring of His195 about the C-C bond. Less favourable hydrogen bonds involving C-H are marked red.

3.2. Possibilities: protonation states of His195 combined with hydrogenated forms of FeMo-co The four possible protonation states of His195 are His-H, His-H, His- and His-HH+: these and their distal connections to HOH532 and Tyr281 are shown in Scheme 2. All four were investigated, as the corresponding modifications of model 1. Each of these isomers was optimised with various representative degrees of relevant hydrogenation of FeMo-co. The total charges on these models were consistent with addition of hydrogen atoms (ie protons + electrons) to FeMo-co and protonation/deprotonation of His195.

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His-H

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His-H

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Scheme 2. The protonation states of His195. Figure 3 (a) shows the pertinent dimensions for eight optimised complete models with His195 configured as His-H. When there is no H atom at the exo position of Fe2 (His-H: 1), the NH atom is directed towards S2B at hydrogen bonding distance (the calculated N--S2B distance is 3.44Å, compare 3.22Å in the crystal structure) and in van der Waals contact with the H atom on S2B. However, with exo-Fe2-H (structures His-H: 2a, His-H: 2b, His-H: 3, His-H: 4), the His195 ring twists such that the NH is directed towards and close to the H atom on Fe2. This important result is emphasised in red on Fig 3 (a). The resultant N-H-H-Fe connection is a dihydrogen bond, to be discussed below. Note that the presence of endo-coordinated H2 on Fe2 or Fe6 does not affect the dihydrogen bond, and that its length is partially dependent on the spin state of FeMo-co. Structures His-H: 5, His-H: 6 and His-H: 7 demonstrate that H2 bound in the exo position of Fe2 does not form a dihydrogen bond with NH, but causes steric displacement of the His195 ring such that the hydrogen bond from NH to S2B is elongated (H--S2B = 2.91, 2.79, 3.13Å) towards non-existence.

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Fig. 3. Optimised structures for the complete model 1 in which the four protonation states of His195 are combined with different forms of hydrogenated FeMo-co: the diagrams show only the relevant contact between His195 and FeMo-co, except for His– : 1 and His– : 2 where the hydrogen atoms on W531 and its hydrogen bonding connections are included. Unmarked spin states are HisH: 1 S=3/2, His-H: 3 S=0, His-H: 4 S=1, His-H: 5 S=1/2, His-H: 6 S=2, His-H: 7 S=1, His– : 1 S=1, His– : 2 S=1/2, His-HH+ : 1 S=1/2, His-HH+ : 2 S=1.

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Structures with the alternative His-H form of His195 are shown in Fig 3 (b). No dihydrogen bond can form, but the presence of H at exo-Fe2 affects the positioning of His195. The only possible hydrogen bond is between S2B-H and N, but this very long and bent (100°) in His-H: 1, and slightly better (2.57Å, 126°) in His-H: 2. The distant interaction between exo-Fe2-H and N in His-H: 2 is not a potential hydrogen bond because the His195 ring is not directed towards exo-Fe2-H.

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When His195 is deprotonated (and HOH531 is configured to be unable to reprotonate), as in models His– : 1 and His– : 2 (Fig 3 (c)), possible hydrogen bond acceptance from S2B-H is long and bent (127°) even though the imidazole ring is directed approximately towards S2B. A shorter interaction occurs between the atoms of H2 coordinated at the exo position of Fe2 (model His– : 2) and the geometry is indicative of an Fe-H(H)--N interaction akin to a hydrogen bond. When His195 is protonated there is strengthening of interactions involving NH. In the absence of exoFe2-H (model His-HH: 1, Fig 3 (d)) the hydrogen bond to S2B is strong (H-S 2.21Å, N-H-S 176°) and enhanced relative to the same interaction in model His-H: 1. With exo-Fe2-H the dihydrogen bond is formed (model His-HH: 2, red emphasis) and is shorter (H-H 1.39Å) than the corresponding dihydrogen bond that occurs when His195 is not protonated (model His-H: 2).

3.3. Dihydrogen bonding Dihydrogen bonding X-H•••H-Y,[69, 70, 71, 72, 73] also described as protonic-hydridic bonding to reflect the polarisation,[74, 75] was recognised first in 1994 as an intramolecular Ir-H--H-N interaction in organometallic compounds,[76, 77, 78] and soon thereafter was observed as intermolecular interactions between heterocyclic amines and metal polyhydride complexes.[79, 80] Experimental and theoretical investigations indicate that the strength of dihydrogen bonding ranges 3 - 7 kcal mol-1,[69, 81, 82, 83, 84, 85] or greater with stronger proton-donating components.[86, 87]

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Dihydrogen bonding is proposed to contribute to (a) the protonation of metal hydride complexes,[88, 89, 90] (b) coordinated dihydrogen functioning as proton donor,[91] (c) the reactions of complexes mimicking hydrogenase enzymes,[92, 93] and (d) metal complexes functioning as electrocatalysts for heterolytic reactions of H2.[94]

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Figure 4 depicts three dihydrogen bonds with accurate experimental structures (A and B are by neutron diffraction). The cationic complex C was crystallised with BF4–, and when calculated without these anions the H---H separation contracts from 1.88 to 1.40Å.[92] The 1.49Å separation of H atoms in A demonstrates the tightness of dihydrogen bonding. Inverse correlation between the acidity of the protonic component and the (calculated) length of the dihydrogen bond, as in His-H: 2 and His-HH: 2 above, has been found in other systems where the H--H separations extend down to 1.37Å,[73, 87] comparable with 1.39Å in His-HH: 2.

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Fig. 4. Accurate experimental structures of dihydrogen bonds. A[95] and B[79, 96] are results from neutron diffraction. Structure C is from ref [92]. 3.4. Formation of exo-Fe2-H2 via dihydrogen bonding with His195

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The short dihydrogen bond in His-H: 2 (and other structures in Fig 3) suggests H atom transfer between His195 and Fe2, as in Scheme 3. There is precedent for this reversible reaction type in model systems, in which M-H complexes form M-H2 when treated with acids,[73, 87, 88, 90, 97, 98] and coordinated H2 can function as proton donor.[91, 97] Therefore, the two reactions shown in Scheme 3 have been simulated, within model 1. These involve partially hydrogenated FeMo-co interacting with protonated or unprotonated His195. Note that the H atoms on FeMo-co have been formed by the coupled protonation and electronation,[7, 99] that generate the EHn intermediates in the Thorneley-Lowe mechanistic framework.[35] The EH3 intermediate structure used here, with H atoms on Fe6, S2B and Fe2, was selected because it is energetically favourable, and because H atom migration from S3B populates Fe6 and S2B before Fe2.[99, 100]

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Scheme 3. Abbreviated representations of two reactions forming H2 on Fe2.

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In reaction (1), with FeMo-co in its normal EH3 redox level and with three added H atoms, the relative potential energies of the reactant HN: NH, the transition state TS1, and the H2 complex HN: FeH2 are 0, +5 and -7 kcal mol-1 for the S=0 spin state of FeMo-co, and 0, +3.5 and -11 kcal mol-1 in the S=1 spin state. The geometry at the transition state has N-H = 1.35Å (S=0), 1.31Å (S=1), which is closer to the reactant geometry (N-H = 1.08Å), consistent with the small barriers of ca 4 kcal mol-1. The dihydrogen bond N-H--H-Fe exists in the reactant HN: NH. Structure His-HH+ : 2 shown in Fig 3 (d) with a dihydrogen bond distance H--H = 1.39Å is the same as the reactant HN: NH (S=1) in reaction (1): the dihydrogen bond shortens just slightly to 1.35Å in the transition state. These results for reaction (1) were obtained with a dielectric constant  of 5 in the solvation model: with  = 15 and S=1 the energy profile differs very slightly, with TS1 and HN: FeH2 at +5.0 and -9 kcal mol-1 respectively. Reaction (1) was then explored for a one electron reduced state, with S=1/2. The equilibrium is biased towards the HN: FeH2 product, with an overall potential energy change of -14 kcal mol-1, and a transition state that is very close to the reactant. This suggests that the additional negative charge is located more on FeMo-co that His195, and that the Fe-H moiety in the reactant is more basic, as expected. Reaction (2), devoid of N-H, was then simulated. In the normal EH3 redox level (and S=0), the equilibrium is biased towards the NH reactant, and the transition state is very close to FeH2. The lack of precision here occurs because exploration for the transition states in reactions (1) and (2) is complicated by the number of variables (the locations of N and of two H atoms relative to Fe2, ie the N-H, H-H, HFe, H-Fe and N--Fe distances) and the relatively flat saddle domain. There is an additional complication, because the binding of H2 to Fe2 is weak (further information below) and in some simulations the product H2 is uncoordinated. The net potential energy change for reaction (2) at this normal redox level is ca -12 kcal mol-1. Reaction (2) with one additional electron (and S=1/2) has a better-defined transition state, 11 kcal mol-1 above the reactant, with an N-H distance of 1.54Å, and H--H 0.89Å. The net potential energy

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change for formation of FeH2 is ca +7 kcal mol-1: the potential energy change for the subsequent dissociation of H2 is ca -3 kcal mol-1.

3.5. Association/dissociation of Fe2-H2

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In summary, the results for reactions (1) and (2), each in two charge/redox states, substantiate the expected qualitative interpretation that (a) the absence of N-H on His195 increases its basicity and shifts the equilibrium towards formation of N-H, (b) the presence of N-H on His195 increases its acidity and shifts the equilibrium towards FeH2, and (c) electron injection increases the basicity of the FeMo-co species and shifts the equilibrium towards H2 formation. The potential energy surface is relatively flat, without prohibitive barriers in either direction. Reaction (1) at the normal EH3 level is the most favourable of the four simulated.

The reactions just described form H2 by N-H approaching exo-Fe2-H, but in a trajectory that presents H2 in a geometry between end-on and side-on to Fe (ie one Fe-H short and one long), which must twist in order to achieve the 2-H2 binding geometry. Some trajectory simulations, mainly for reaction (2), formed unbound H2 which subsequently diffused to a large cavity bounded by His195, Ser278, S2A and Fe2, with a downhill energy of 5 – 6 kcal mol-1 as the Fe2--H2 separation increased to ca 3.4Å.

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For the Fe-H2 product of reaction (1) (HN: FeH2) the potential energy barrier for dissociation of H2 is calculated to be 2 kcal mol-1, while the barrier for association (from an arbitrary asymmetric location with Fe-H distances 2.6, 2.9Å) is 2.5 kcal mol-1. These barriers are smaller than those calculated earlier using models without the enclosing protein components.[62] 3.6. H transfer between S2B and His195

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The feasibility of H atom transfer between S2B and N of His195 was tested, in reactions (3) with N-H and (4) without N-H (Scheme 4). In both reactions there is a preparatory phase, in which the H-S2B bond bends towards N, prior to H transfer For reaction (3) [S = 0] the S2B-H--N bond angle starts at 106°, then adjusts to 140° with an energy penalty of 6.5 kcal mol-1, and then proceeds to TS3 (S2B--H-N 160°) with a further energy increase of 5 kcal mol-1. The energy decrease from TS3 to the product is 9.5 kcal mol-1, and the net energy change for reaction (3) is + 2 kcal mol-1. The effect of addition of one electron to the system was tested for reaction (3) [S = 1/2 throughout]. The barrier from reactant HN: S2BH to TS3 increases slightly from 11.5 to 13 kcal mol-1, and the net energy change is + 6 kcal mol-1.

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Scheme 4. Abbreviated representations of two reactions for H transfer between S2B and His195.

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For reaction (4) without N-H, at the normal redox level with S=0, the barrier to TS4 is 7 kcal mol-1, and the overall energy change is -21 kcal mol-1. On reduction by one electron with S=1/2, the energies of transition state TS4 and product NH relative to reactant S2BH are +13 and -8 kcal mol-1. The dependence of the reaction energetics on the N protonation state of His195 and the degree of electronation of FeMo-co is presented in Table 1. Transfer of H from S2B has larger barrier when the electron population is increased, and the absence of N-H, eq (4), results in a more negative energy change for formation of N-H, as expected. Table 1. Calculated reaction energies and transition barriers for H transfer between S2B and His195 (reactions (3) and (4)) for two electron populations. standard redox level barriers (kcal mol-1) S2B-H  N N-H  S2B 11.5 9.5 7 28

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one electron added barriers (kcal mol-1) S2B-H  N N-H  S2B 13 7 13 21

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The N protonation state of His195 and the degree of electronation of FeMo-co are reflected also in the position of H along the reaction coordinate between S2B and N for the four transition states. The distances are contained in Table 2. As expected, the position of H is further from S2B when FeMo-co is electronated, reflecting slightly increased basicity of S2B on electronation, and the position of H is further from N in reaction (4), reflecting the larger basicity of the His side-chain when not protonated at N. Table 2. Dimensions (Å) of the transition states for reactions (3) and (4), each at two degrees of electronation of FeMo-co. reaction (3) (4)

standard redox level H--S2B H--N 1.56 1.44 1.45 1.88

one electron added H--S2B H--N 1.64 1.35 1.51 1.62

The four calculated energy barriers for H transfer from S2B to N of His195 range from 7 to 13 kcal mol1, indicating that this process is feasible. The reverse reaction, hydrogenation of S2B by transfer from N of His195 is also feasible when N-H is present, reaction (3).

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3.7. Involvement of HOH531

(a)

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Reactions (2) and (4) involved deprotonated His195, with neither N-H nor N-H, and a formal negative charge. The simulations just described included the arrangement of H atoms on HOH531 and Tyr281 shown in Scheme 5 (a). In this configuration the water molecule is not able to protonate the imidazolate– ring, because the OH– that would be generated is more basic than imidazolate–. Thus the preceding simulations of reactions (2) and (4) were not complicated by involvement of HOH531. However, if the H atoms on HOH531 are directed as shown on the left side of Scheme 5 (b), simulations show that a proton is transferred from the phenol function of Tyr281 to HOH531, concomitant with protonation of N by HOH531. This two-stage proton transfer reflects greater basicity of imidazolate– than of phenolate– in this context, with HOH531 as the proton transfer intermediary.

(b) Scheme 5. Configurations of hydrogen atoms on HOH531, in relation to His195 and Tyr281. Reaction (5) involves the same transfer of H between S2B and N of His195 as does reaction (4), but instead has HOH531 oriented such that it can mediate proton transfer from Tyr281. This reaction involving three H atom transfers (emphasised in red on equation (5)) has been simulated with protein model 1: there is one transition state (TS5). In the normal redox level of FeMo-co (here starting as an EH2 intermediate, S=0), the relative potential energies are HOH: S2BH 0, TS5 +16, HOH: NH -9 kcal mol-1. On reduction of the complete system by one electron (S=1/2) the relative energies are HOH: S2BH 0, TS5 +19, HOH: NH +3 kcal mol-1. These results also support the possibility of reversible H-transfer between S2B and N of His195.

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3.8. Atom partial charges

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Scheme 6. Abbreviated representation of H-transfer between Tyr281 and S2B, mediated by HOH531 and His195.

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Atom partial charges have been calculated for the structures and reactions described above, in order to understand the distribution of charge, its variation with overall electron reduction, and charge changes during the reactions. Complete results are provided in the Supplementary Information. The principal results can be understood from the data in Table 3, namely the Mulliken partial charges for selected atoms of the reactants and products of reaction (3), at two levels of electronation. (a) The Fe atoms of FeMo-co have charges, ranging +0.40 to +0.60, that are invariant through the reaction and with addition of one electron. (b) The negative charge on the central atom Cc is invariant. (c) The S atoms of FeMo-co have charges in the range -0.45 to -0.87, and bear most of the additional negative charge when an electron is added to the total model. (d) The H atom bound to Fe6 has a very small negative charge (-0.03 to -0.12) that hardly changes during the reaction, or on addition of one electron. (e) The H atom that transfers from S2B to N of His195 during the reaction has a small positive charge (+0.23 to +0.30) that hardly changes during the reaction, and is unchanged by addition of one electron. (f) The charge on S2B becomes more negative (by ca 0.25) when it loses its H atom to N of His195 during the reaction. (g) The charge of N of His195 becomes correspondingly less negative when it gains the H atom from S2B. (h) Charges on the more remote atoms of His195, HOH531 and Tyr281 are invariant. Analogous results for reactions (1) and (5) are tabulated in the Supplementary Information. In reaction (1) the atoms from His195 and Fe2 that are dihydrogen bonded and then form H2 bound to Fe2 have small charges in the range -0.18 to +0.26. Overall, the appropriate conceptual frame for the various interactions of His195 with hydrogenated FeMoco is one of low bond polarity and H atom transfer steps.

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Table 3. Mulliken partial charges calculated for selected atoms in reaction (3). The significant values are emboldened. reaction (3) reaction (3) + electron HNδ: S2BH S=0 HNd: NeH S=0 HNδ: S2BH S=1/2 HNδ: NεH S=1/2 atom name charge atom name charge atom name charge atom name charge Cc -0.73 Cc -0.72 Cc -0.72 Cc -0.72 Fe1 0.60 Fe1 0.60 Fe1 0.60 Fe1 0.60 Fe2 0.56 Fe2 0.60 Fe2 0.56 Fe2 0.62 Fe4 0.56 Fe4 0.56 Fe4 0.58 Fe4 0.59 Fe3 0.57 Fe3 0.58 Fe3 0.56 Fe3 0.57 Fe5 0.52 Fe5 0.53 Fe5 0.55 Fe5 0.57 Fe6 0.40 Fe6 0.44 Fe6 0.40 Fe6 0.45 Fe7 0.59 Fe7 0.59 Fe7 0.57 Fe7 0.58 S2BH S2BH 0.24 0.29 0.23 0.30 H-His195 H-His195 H-HOH531 0.34 H-HOH531 0.35 H-HOH531 0.34 H-HOH531 0.35 0.26 0.31 0.26 0.30 H-His195 H-His195 H-His195 H-His195 H-HOH531 0.29 H-HOH531 0.30 H-HOH531 0.29 H-HOH531 0.30 H(O)-Tyr281 0.30 H(O)-Tyr281 0.31 H(O)-Tyr281 0.30 H(O)-Tyr281 0.31 Fe6H -0.03 Fe6H -0.07 Fe6H -0.07 Fe6H -0.12 -0.33 -0.30 -0.33 -0.30 N-His195 N-His195 N-His195 N-His195 -0.41 -0.28 -0.39 -0.28 N-His195 N-His195 N-His195 N-His195 O(H)-Tyr281 -0.55 O(H)-Tyr281 -0.56 O(H)-Tyr281 -0.55 O(H)-Tyr281 -0.56 O-HOH531 -0.58 O-HOH531 -0.58 O-HOH531 -0.58 O-HOH531 -0.59 S-cys275 -0.67 S-cys275 -0.69 S-cys275 -0.71 S-cys275 -0.72 S1A -0.52 S1A -0.59 S1A -0.63 S1A -0.70 S1B -0.50 S1B -0.51 S1B -0.56 S1B -0.59 S2A -0.58 S2A -0.63 S2A -0.62 S2A -0.67 S2B S2B S2B S2B -0.45 -0.68 -0.48 -0.74 S3A -0.63 S3A -0.68 S3A -0.77 S3A -0.83 S3B -0.56 S3B -0.59 S3B -0.60 S3B -0.62 S4A -0.58 S4A -0.64 S4A -0.68 S4A -0.74 S4B -0.53 S4B -0.54 S4B -0.59 S4B -0.62 S5A -0.77 S5A -0.81 S5A -0.84 S5A -0.87

4. Discussion This paper presents density functional calculations using molecular systems extending into the relevant protein surrounding the active site FeMo-co, and thereby able to provide direct quantum evaluation of reactions involving bond-making and bond-breaking between FeMo-co and its surrounds. These simulations have defined ways in which the experimentally significant amino acid His195 can participate in reactions at the catalytic domain of FeMo-co. The capacity of this residue to hydrogen bond with S2B was evident in the first crystal structure, but the possibility that the imidazole ring of His195 could donate a proton to substrate or intermediate bound to FeMo-co has previously been speculative. The ability of the side-chain of His195 to exchange H atoms with S2B has now been characterised, with potential energy barriers ranging 7 to 28 kcal mol-1 depending on the protonation state of the His195 side chain and the redox level of the protein. The side chain of His195 can also be directed towards the exo-position of Fe2, considered to be a key site in the overall mechanism, and is able to transfer an H atom to exo-Fe2-H, Dance_His195_reactions: JINORGBIO_2016_4 revised

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generating H2, or to extract an H atom from H2 bound in the exo position of Fe2. Also, participation of the water molecule on the distal side of the His195 side-chain has been described, and its capacity to shuttle a proton from the side-chain of Tyr228 when needed has been quantitated. These reactions of His195 do not require movement of the adjacent residues, or of the helix in which it is located.

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Considerable flapping movement of the side-chain imidazole group of His195 occurs in these simulations of its reactivity. However, the idea that the side-chain imidazole could rotate through ca 180° about its C-C bond, and thereby interchange its N atoms and facilitate serial transfer of protons from the surface of the protein to S2B of FeMo-co, is shown to be untenable for reasons of geometry. The molecular dynamics study by Smith et al[101] mentions that His195 can approach S2A, with H--S2A = 2.6Å, but such movement was not evident in the simulations undertaken here.

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These simulations of the reactivity of His195 suggest that it could catalyse the formation and elimination of H2. Notice that reaction (1), followed by dissociation of H2, and then reaction (3) effects the conversion of two hydrogen atoms on FeMo-co, namely S2B-H plus exo-Fe2-H, to H2. This sequence, in which His195 is functioning as catalyst, is shown in Scheme 7. These reactions bypass the formation of H2 by direct combination of S2B-H and Fe2-H. The barrier energies, marked on Scheme 7, suggest that this a feasible process in the mechanistic fabric of nitrogenase.

Scheme 7. The sequence of reactions whereby His195 (green) catalyses the formation and elimination of H2 from H atoms on S2B and Fe2 (yellow). The reaction barrier energies in kcal mol-1 are marked. Dance_His195_reactions: JINORGBIO_2016_4 revised

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The next stage of this investigation is assessment of the ability of protonated His195 to transfer a proton to bound N2. In the series of hydrogenations of N2 leading to 2NH3, the first, forming N2H, is the most difficult. All mechanistic proposals for this step have used an H atom or atoms already located on FeMoco. However, since protonated His195 is acidic and demonstrably able to transfer a proton, and because the His195 side-chain can sufficiently approach N2 in some of its binding modes at Fe2 and/or Fe6, direct transfer from His195 to N2 needs to be investigated using the enlarged density functional model. It is expected that this step would be favoured by prior or concurrent electron-transfer to FeMo-co, increasing the basicity of bound N2: this expectation is supported by the results reported in this work for His195 as a proton donor to H-Fe2 or S2B, a step facilitated by addition of one electron. The hypothesis is that His195 functions only as the agent for the first hydrogenation of bound N2, and that subsequent H atom transfers to N2 use H atoms bound to FeMo-co. This proposal, coupled with the reasonable hypothesis that the initial binding of N2 occurs with a favourable location and geometry created by the dissociation of H2, is compatible with the experimental data of Hoffman et al[34] on N2/H2 exchange. The associative or dissociative character of the reversible N2/H2 exchange is still an open question.[102]

Acknowledgments

Supplementary information

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This research is supported by UNSW Australia, and was undertaken with the assistance of resources provided at the NCI National Facility at the Australian National University through the National Computational Merit Allocation Scheme supported by the Australian Government.

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Validation of computational procedures (S1), Cartesian coordinates for reported structures (Table S2), Fe2-Cc and Fe6-Cc distance variations (Table S3), atom partial charges (spreadsheet), comparative atom charges (Tables S4, S5, S6).

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References [1]

B. K. Burgess, D. J. Lowe, Chem. Rev. 96 (1996) 2983-3011.

[2]

J. Christiansen, D. R. Dean, L. C. Seefeldt, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52 (2001)

T

269-295. R. Y. Igarashi, L. C. Seefeldt, Crit. Rev. Biochem. Mol. Biol. 38 (2003) 351-384.

[4]

D. C. Rees, F. A. Tezcan, C. A. Haynes, M. Y. Walton, S. Andrade, O. Einsle, J. A. Howard, Phil.

MA NU SC R

IP

[3]

Trans. Roy. Soc. A 363 (2005) 971-984.

J. B. Howard, D. C. Rees, Proc. Nat. Acad. Sci. USA 103 (2006) 17119-17124.

[6]

L. C. Seefeldt, B. M. Hoffman, D. R. Dean, Annu. Rev. Biochem. 78 (2009) 701-722.

[7]

I. Dance, Chem Commun (Camb) 49 (2013) 10893-10907.

[8]

B. M. Hoffman, D. Lukoyanov, Z.-Y. Yang, D. R. Dean, L. C. Seefeldt, Chemical Reviews 114 (2014) 4041-4062.

TE D

[5]

I. Dance, Zeitschrift für anorganische und allgemeine Chemie 641 (2015) 91-99.

[10]

O. Einsle, F. A. Tezcan, S. L. A. Andrade, B. Schmid, M. Yoshida, J. B. Howard, D. C. Rees,

CE P

[9]

Science 297 (2002) 1696-1700. T. Spatzal, M. Aksoyoglu, L. Zhang, S. L. A. Andrade, E. Schleicher, S. Weber, D. C. Rees, O.

AC

[11]

Einsle, Science 334 (2011) 940. [12]

D. C. Rees, M. K. Chan, J. Kim, Adv. Inorg. Chem. 40 (1993) 89-119.

[13]

J. B. Howard, D. C. Rees, Chem. Rev. 96 (1996) 2965-2982.

[14]

I. Dance, in Bioinspired Catalysis: Metal-Sulfur Complexes (Eds.: W. Weigand, P. Schollhammer), Wiley-VCH, Weinheim, Germany, 2015, pp. 249-288.

[15]

L. C. Seefeldt, I. G. Dance, D. R. Dean, Biochemistry 43 (2004) 1401-1409.

[16]

B. M. Barney, R. Y. Igarashi, P. C. Dos Santos, D. R. Dean, L. C. Seefeldt, J. Biol. Chem. 279 (2004) 53621-53624.

Dance_His195_reactions: JINORGBIO_2016_4 revised

January 4, 2017

Page 22 of 29

[17]

ACCEPTED MANUSCRIPT

P. C. Dos Santos, S. M. Mayer, B. M. Barney, L. C. Seefeldt, D. R. Dean, Journal of Inorganic Biochemistry 101 (2007) 1642-1648.

[18]

H. Thomann, M. Bernardo, W. E. Newton, D. R. Dean, Proceedings of the National Academy of Sciences 88 (1991) 6620-6623. C. H. Kim, W. E. Newton, D. R. Dean, Biochem. 34 (1995) 2798-2808.

[20]

M. J. Dilworth, K. Fisher, C. H. Kim, W. E. Newton, Biochemistry 37 (1998) 17495-17505.

[21]

K. Fisher, M. J. Dilworth, W. E. Newton, Biochemistry 39 (2000) 15570-15577.

[22]

K. Fisher, M. J. Dilworth, C.-H. Kim, W. E. Newton, Biochemistry 39 (2000) 10855-10865.

[23]

K. Fisher, M. J. Dilworth, C.-H. Kim, W. E. Newton, Biochemistry 39 (2000) 2970-2979.

[24]

M. Sorlie, J. Christiansen, B. J. Lemon, J. W. Peters, D. R. Dean, Biochemistry 40 (2001) 1540-

MA NU SC R

IP

T

[19]

[25]

TE D

1549.

B. M. Barney, D. Lukoyanov, T.-C. Yang, D. R. Dean, B. M. Hoffman, L. C. Seefeldt, Proc. Nat. Acad. Sci. USA 103 (2006) 17113-17118. K. Fisher, D. J. Lowe, P. Tavares, A. S. Pereira, B. H. Huynh, D. Edmondson, W. E. Newton, J.

CE P

[26]

Inorg. Biochem. 101 (2007) 1649-1656. Z.-Y. Yang, K. Danyal, L. C. Seefeldt, Methods Mol. Biol. 766 (2011) 9-29.

[28]

P. M. C. Benton, M. Laryukhin, S. M. Mayer, B. M. Hoffman, D. R. Dean, L. C. Seefeldt,

AC

[27]

Biochemistry 42 (2003) 9102-9109. [29]

H.-I. Lee, R. Igarashi, M. Laryukhin, P. E. Doan, P. C. Dos Santos, D. R. Dean, L. C. Seefeldt, B. M. Hoffman, J. Am. Chem. Soc. 126 (2004) 9563-9569.

[30]

R. Igarashi, P. C. Dos Santos, W. G. Niehaus, I. G. Dance, D. R. Dean, L. C. Seefeldt, J. Biol. Chem. 279 (2004) 34770-34775.

[31]

I. Dance, J. Am. Chem. Soc. 126 (2004) 11852-11863.

Dance_His195_reactions: JINORGBIO_2016_4 revised

January 4, 2017

Page 23 of 29

[32]

ACCEPTED MANUSCRIPT

P. C. Dos Santos, R. Igarashi, H.-I. Lee, B. M. Hoffman, L. C. Seefeldt, D. R. Dean, Acc. Chem. Res. 38 (2005) 208-214.

[33]

D. Lukoyanov, Z.-Y. Yang, N. Khadka, D. R. Dean, L. C. Seefeldt, B. M. Hoffman, Journal of the American Chemical Society 137 (2015) 3610-3615.

T

D. Lukoyanov, N. Khadka, Z.-Y. Yang, D. R. Dean, L. C. Seefeldt, B. M. Hoffman, Journal of the

[35]

R. N. F. Thorneley, D. J. Lowe, in Molybdenum enzymes (Ed.: T. G. Spiro), Wiley Interscience, New York, 1985, pp. 221-284.

[36]

Z.-Y. Yang, V. R. Moure, D. R. Dean, L. C. Seefeldt, Proceedings of the National Academy of Sciences 109 (2012) 19644-19648.

[37]

MA NU SC R

American Chemical Society 138 (2016) 10674-10683.

IP

[34]

S. Shaw, D. Lukoyanov, K. Danyal, D. R. Dean, B. M. Hoffman, L. C. Seefeldt, Journal of the

TE D

American Chemical Society 136 (2014) 12776-12783. I. Dance, Dalton Transactions 44 (2015) 9027-9037.

[39]

B. Delley, J. Chem. Phys. 92 (1990) 508-517.

[40]

B. Delley, in Modern density functional theory: a tool for chemistry, Vol. 2 (Eds.: J. M.

CE P

[38]

AC

Seminario, P. Politzer), Elsevier, Amsterdam, 1995, pp. 221-254. [41]

J. Baker, A. Kessi, B. Delley, The Journal of Chemical Physics 105 (1996) 192-212.

[42]

B. Delley, J. Chem. Phys. 113 (2000) 7756-7764.

[43]

J. Andzelm, R. D. King-Smith, G. Fitzgerald, Chemical Physics Letters 335 (2001) 321-326.

[44]

I. Dance, CrystEngComm. 5 (2003) 208-221.

[45]

S. Grimme, J. Comput. Chem. 27 (2006) 1787-1799.

[46]

A. D. Becke, Phys. Rev. A 38 (1988) 3098-3100.

[47]

C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 37 (1988) 785-789.

[48]

I. Dance, Inorg. Chem. 50 (2011) 178-192.

Dance_His195_reactions: JINORGBIO_2016_4 revised

January 4, 2017

Page 24 of 29

ACCEPTED MANUSCRIPT

A. Klamt, G. Schüürmann, J. Chem.Soc., Perkin Trans. 2 (1993) 799-805.

[50]

J. Andzelm, C. Kolmel, A. Klamt, J. Chem. Phys. 103 (1995) 9312-9320.

[51]

B. Delley, Molecular Simulation 32 (2006) 117-123.

[52]

I. Dance, Molecular Simulation 34 (2008) 923-929.

[53]

I. Dance, Molecular Simulation 37 (2011) 257.

[54]

I. Dance, Dalton Transactions 44 (2015) 18167-18186.

[55]

R. S. Mulliken, J. Chem. Phys. 23 (1955) 1833-1846.

[56]

F. L. Hirshfeld, Theoret. Chim. Acta 44 (1977) 129-138.

[57]

I. G. Dance, in Transition Metal Sulfur Chemistry: Biological and Industrial Significance, Vol.

MA NU SC R

IP

T

[49]

653 (Eds.: E. I. Stiefel, K. Matsumoto), American Chemical Society, Washington, DC, USA,

TE D

1996, pp. 135-152.

I. Dance, J. Chem. Soc., Chem. Commun. (1998) 523-530.

[59]

Z. X. Cao, X. Jin, Q. N. Zhang, Journal of Theoretical and Computational Chemistry 4 (2005) 593-602.

CE P

[58]

I. Dance, Dalton Trans. 41 (2012) 4859-4865.

[61]

C. Bianchini, D. Masi, M. Peruzzini, M. Casarin, C. Maccato, G. A. Rizzi, Inorg. Chem. 36

AC

[60]

(1997) 1061-1069. [62]

I. Dance, J. Am. Chem. Soc. 127 (2005) 10925-10942.

[63]

I. Dance, J. Am. Chem. Soc. 129 (2007) 1076-1088.

[64]

L. B. Gee, I. Leontyev, A. Stuchebrukhov, A. D. Scott, V. Pelmenschikov, S. P. Cramer, Biochemistry 54 (2015) 3314-3319.

[65]

C. N. Morrison, J. A. Hoy, L. Zhang, O. Einsle, D. C. Rees, Biochemistry 54 (2015) 2052-2060.

[66]

S. J. George, B. M. Barney, D. Mitra, R. Y. Igarashi, Y. Guo, D. R. Dean, S. P. Cramer, L. C. Seefeldt, Journal of Inorganic Biochemistry 112 (2012) 85-92.

Dance_His195_reactions: JINORGBIO_2016_4 revised

January 4, 2017

Page 25 of 29

[67]

ACCEPTED MANUSCRIPT

A. D. Scott, V. Pelmenschikov, Y. Guo, L. Yan, H. Wang, S. J. George, C. H. Dapper, W. E. Newton, Y. Yoda, Y. Tanaka, S. P. Cramer, Journal of the American Chemical Society 136 (2014) 15942-15954.

[68]

M. Maiuri, I. Delfino, G. Cerullo, C. Manzoni, V. Pelmenschikov, Y. Guo, H. Wang, L. B. Gee,

IP

T

C. H. Dapper, W. E. Newton, S. P. Cramer, Journal of Inorganic Biochemistry 153 (2015) 128-

[69]

MA NU SC R

135.

R. H. Crabtree, P. E. M. Siegbahn, O. Eisenstein, A. L. Rheingold, T. F. Koetzle, Acc. Chem. Res. 29 (1996) 348-354.

R. Custelcean, J. E. Jackson, Chemical Reviews 101 (2001) 1963-1980.

[71]

L. M. Epstein, E. S. Shubina, Coord. Chem. Rev. 231 (2002) 165-181.

[72]

N. V. Belkova, E. S. Shubina, L. M. Epstein, Accounts of Chemical Research 38 (2005) 624-631.

[73]

N. V. Belkova, L. M. Epstein, O. A. Filippov, E. S. Shubina, Chemical Reviews 116 (2016) 8545-

TE D

[70]

8587.

A. J. Lough, S. Park, R. Ramachandran, R. H. Morris, Journal of the American Chemical Society

CE P

[74]

116 (1994) 8356-8357.

K. Abdur-Rashid, A. J. Lough, R. H. Morris, Canadian Journal of Chemistry 79 (2001) 964-976.

[76]

S. Park, R. Ramachandran, A. J. Lough, R. H. Morris, Journal of the Chemical Society, Chemical

AC

[75]

Communications (1994) 2201-2202. [77]

J. C. Lee, A. L. Rheingold, B. Muller, P. S. Pregosin, R. H. Crabtree, Journal of the Chemical Society, Chemical Communications (1994) 1021-1022.

[78]

J. C. Lee, E. Peris, A. L. Rheingold, R. H. Crabtree, Journal of the American Chemical Society 116 (1994) 11014-11019.

[79]

J. Wessel, J. C. Lee, E. Peris, G. P. A. Yap, J. B. Fortin, J. S. Ricci, G. Sini, A. Albinati, T. F. Koetzle, O. Eisenstein, A. L. Rheingold, R. H. Crabtree, Angewandte Chemie International Edition in English 34 (1995) 2507-2509.

Dance_His195_reactions: JINORGBIO_2016_4 revised

January 4, 2017

Page 26 of 29

[80]

ACCEPTED MANUSCRIPT

B. P. Patel, W. Yao, G. P. A. Yap, A. L. Rheingold, R. H. Crabtree, Chemical Communications (1996) 991-992. Q. Liu, R. Hoffmann, Journal of the American Chemical Society 117 (1995) 10108-10112.

[82]

I. Alkorta, I. Rozas, J. Elguero, Chemical Society Reviews 27 (1998) 163-170.

[83]

G. Orlova, S. Scheiner, T. Kar, J. Phys. Chem. A 103 (1999) 514-520.

[84]

F. Maseras, A. Lledos, E. Clot, O. Eisenstein, Chem. Rev. 100 (2000) 601-636.

[85]

Natalia V. Belkova, Ekaterina V. Bakhmutova, Elena S. Shubina, C. Bianchini, M. Peruzzini,

MA NU SC R

IP

T

[81]

Vladimir I. Bakhmutov, Lina M. Epstein, European Journal of Inorganic Chemistry 2000 (2000) 2163-2165. [86]

E. V. Bakhmutova, V. I. Bakhmutov, N. V. Belkova, M. Besora, L. M. Epstein, A. Lledós, G. I. Nikonov, E. S. Shubina, J. Tomàs, E. V. Vorontsov, Chemistry – A European Journal 10 (2004)

[87]

TE D

661-671.

N. V. Belkova, E. Collange, P. Dub, L. M. Epstein, D. A. Lemenovskii, A. Lledós, O. Maresca, F.

CE P

Maseras, R. Poli, P. O. Revin, E. S. Shubina, E. V. Vorontsov, Chemistry – A European Journal 11 (2005) 873-888.

J. S. Ricci, T. F. Koetzle, M. T. Bautista, T. M. Hofstede, R. H. Morris, J. F. Sawyer, J. Am.

AC

[88]

Chem. Soc. 111 (1989) 8823-8827. [89]

L. D. Field, T. W. Hambley, B. C. K. Yau, Inorganic Chemistry 33 (1994) 2009-2017.

[90]

J. A. Ayllón, C. Gervaux, S. Sabo-Etienne, B. Chaudret, Organometallics 16 (1997) 2000-2002.

[91]

N. K. Szymczak, L. N. Zakharov, D. R. Tyler, Journal of the American Chemical Society 128 (2006) 15830-15835.

[92]

T. B. Rauchfuss, Accounts of Chemical Research 48 (2015) 2107-2116.

[93]

D. Schilter, J. M. Camara, M. T. Huynh, S. Hammes-Schiffer, T. B. Rauchfuss, Chemical Reviews 116 (2016) 8693-8749.

Dance_His195_reactions: JINORGBIO_2016_4 revised

January 4, 2017

Page 27 of 29

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[94]

R. M. Bullock, M. L. Helm, Accounts of Chemical Research 48 (2015) 2017-2026.

[95]

T. Liu, X. Wang, C. Hoffmann, D. L. DuBois, R. M. Bullock, Angewandte Chemie International Edition 53 (2014) 5300-5304.

[96]

B. P. Patel, J. Wessel, W. B. Yao, J. C. Lee, E. Peris, T. F. Koetzle, G. P. A. Yap, J. B. Fortin, J.

IP

T

S. Ricci, G. Sini, A. Albinati, O. Eisenstein, A. L. Rheingold, R. H. Crabtree, New J. Chem. 21

MA NU SC R

(1997) 413-421. [97]

P. G. Jessop, R. H. Morris, Coord. Chem. Rev. 121 (1992) 155-284.

[98]

L. D. Field, H. L. Li, B. A. Messerle, R. J. Smernik, P. Turner, Dalton Trans. (2004) 1418-1423.

[99]

I. Dance, Inorg Chem 52 (2013) 13068-13077.

[100] I. Dance, Biochemistry 45 (2006) 6328-6340.

TE D

[101] D. Smith, K. Danyal, S. Raugei, L. C. Seefeldt, Biochemistry 53 (2014) 2278-2285. [102] D. Lukoyanov, N. Khadka, Z.-Y. Yang, D. R. Dean, L. C. Seefeldt, B. M. Hoffman, Journal of the

AC

CE P

American Chemical Society 138 (2016) 1320-1327.

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ACCEPTED MANUSCRIPT New insights into the reaction capabilities of His195 adjacent to the active site of nitrogenase Ian Dance

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School of Chemistry, UNSW Australia, 2052

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[email protected]

Highlights

Histidine195 and can react with the active site FeMo-co of the enzyme nitrogenase. A dihydrogen bond can form between His195 and an H-Fe bond of FeMo-co. FeH2 on FeMo-co can be formed by His195. An imidazole ring-flip by His195 is not able to facilitate transfer of protons to FeMo-co. An H atom can transfer between an S atom of FeMo-co and the side-chain of His195.

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