Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase

Accepted Manuscript Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase

Margareta R.A. Blomberg, Pia Ädelroth PII: DOI: Reference:

S0005-2728(18)30644-3 doi:10.1016/j.bbabio.2018.09.368 BBABIO 47970

To appear in:

BBA - Bioenergetics

Received date: Revised date: Accepted date:

3 May 2018 23 August 2018 17 September 2018

Please cite this article as: Margareta R.A. Blomberg, Pia Ädelroth , Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase. Bbabio (2018), doi:10.1016/j.bbabio.2018.09.368

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Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - a comparison between nitric oxide reductase and cytochrome c oxidase. b ¨ Margareta R.A. Blomberg∗a and Pia Adelroth

(a) Department of Organic Chemistry and (b) Department of Biochemistry and Biophysics,

Contact information for corresponding author:

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E-mail: [email protected]. Phone: +46-8-16 26 16

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Arrhenius Laboratory, Stockholm University, SE-106 91, Stockholm, Sweden.

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Abstract Cytochrome c oxidases (CcO) reduce O2 to H2 O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of CcO can also reduce NO to N2 O and water while others cannot. Here, the mechanism for NO-reduction in CcO is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction

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in a “true” cytochrome c-dependent NO reductase (cNOR). The calculations show that in cNOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced,

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while the higher reduction potentials in CcO lead to a slower or even impossible reaction, consistent with experimental observations. In both enzymes the reaction is initiated by

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addition of two NO molecules to the reduced active site, forming a hyponitrite intermediate. In cNOR, N2 O can then be formed using only the active-site electrons. In contrast, in CcO,

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one proton-coupled reduction step most likely has to occur before N2 O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different CcO species with the same heme a3 -Cu active site differ with respect to NO reduction

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efficiency, since they have a varying number and/or properties of proton channels. Finally, the calculations also indicate that a conserved active site valine plays a role in reducing the

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rate of NO reduction in CcO.

Keywords: heme-copper oxidases, NO reduction, density functional theory, reduction

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potentials, reaction mechanisms, energy profiles

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1. Introduction The reduction of nitric oxide to nitrous oxide and water according to equation (1) is a key step of denitrification, an anaerobic respiratory process catalyzed by nitric oxide reductases (NORs). The NORs belong to the membrane-bound heme-copper oxidase (HCuO) superfamily of enzymes, which also accommodate cytochrome c oxidases (CcOs), which

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reduce molecular oxygen to water according to equation (2) as the final step in aerobic respiration in mitochondria and many bacteria.

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2NO + 2H+ + 2e− → N2 O + H2 O (1)

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O2 + 4H+ + 4e− → 2H2 O (2)

Both these reactions are highly exergonic, and in the CcOs a significant part of the re-

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leased free energy is stored as an electrochemical gradient over the membrane. An interesting difference between the O2 -reducing and the NO-reducing HCuOs, is that in the best characterised NORs, the c (cytochrome c-oxidising) NORs, there is no such energy stor-

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age. Another interesting observation is that there is some cross-reactivity between the two groups of HCuOs, such that some NORs can reduce molecular oxygen [1, 2, 3, 4], and some

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CcOs can reduce nitric oxide [5, 6, 7].

Energy conservation in CcO occurs via two processes. First, the electrons and protons needed for the chemistry (see equation (2)) are taken from opposite sides of the membrane,

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corresponding to net transfer of charge across the membrane, making the chemistry electrogenic. Second, the chemical reaction is coupled to transfer of protons across the entire

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membrane, referred to as proton pumping, which increases the efficiency of energy conservation in the form of a proton electrochemical gradient. For the cNORs it has been found that there is no proton pumping, and the chemistry is not electrogenic, i.e. the electrons and protons for the chemistry (see equation (1)) are taken from the same side of the membrane. This means that all the exergonicity of equation (1) is lost as heat. For an overview of the reactions in the two types of enzymes, see Fig. 1. The two types of enzymes have similar binuclear active sites, labelled the BNC, consisting of a heme-group and a non-heme metal complex. In cNOR the BNC consists of a heme b3 and a non-heme iron, labelled FeB , and the cofactor responsible for the immediate electron transfer into the BNC is a heme b. In the CcOs the non-heme metal is copper, labelled CuB , and the BNC heme-group varies between different classes of CcOs. The largest groups of CcOs, termed the A and B classes [8], have a heme a3 in the BNC and the immediate electron donor is a heme a or a heme b. 3

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Figure 1: Overview of the overall main reactions in CcO (left) and cNOR (right), with the binuclear active site (BNC) and the other redox-active cofactors indicated.

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In the BNC of the CcOs there is also a redox-active tyrosine which is cross-linked to one of the histidine ligands of CuB , and which has been suggested to play an important role in

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the proton pumping process [9]. Although there are large similarities in the BNC, the Aand B-class of CcOs differ substantially in the surrounding enzymes, including the number and composition of proton pathways leading into the BNC [8, 10, 11, 12]. To explain the difference with regard to energy storage between CcO and cNOR it is necessary to know the detailed reaction mechanisms. The CcOs are well studied (for recent reviews, see [13, 14, 15, 16, 17]) and the basic reaction mechanism for O2 reduction is quite simple, with the O-O bond cleavage occurring in one step directly after oxygen binding to the reduced BNC active site. This process requires four electrons and oxidizes the active site cofactors, and the rest of the catalytic cycle consists of four reduction steps in which the active site is rereduced and two water molecules are formed. Certain aspects of this reaction mechanism, however, still remain to be solved, such as the electronic structure and protonation state of some of the intermediates, the exact exergonicity of some of the 4

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reduction steps and details of the proton pumping mechanism. In contrast, there is no consensus on the reaction mechanism of NO reduction in cNOR, and since this reaction is more complicated, with both the formation of a new covalent bond (N-N) from the two molecules of NO, and the cleavage of another one (N-O) to form the N2 O product (and H2 O), there are several quite different mechanisms suggested, see for example [18]. Computational studies have shown that one of the suggested types of mechanisms, the

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so called cis-b3 mechanism is the most likely one [18, 19], and the calculations could also be used to explain why the NO reduction process in cNOR cannot be electrogenic [20].

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Basically the low reduction potentials of the BNC cofactors in cNOR (0.06 V for heme b3

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and 0.32 V for FeB [21]) together with high proton uptake barriers make the reduction steps rate-limiting and slow already without a gradient across the membrane. If the reaction occurred in an electrogenic way the reduction steps, where protons are taken up, would

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become even slower when the gradient is present, and the overall reaction would presumably become too slow for efficient NO removal [19, 20]. On the other hand the low reduction

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potentials increase the exergonicity of the chemical part of the reaction, and most likely also the rate of the disappearance of the toxic NO molecule, making the actual reduction potentials optimal for the entire catalytic cycle to be as fast as possible [19, 20].

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The results and conclusions for NO reduction in cNOR discussed in the previous paragraph imply that the corresponding reaction in CcO would be significantly slower, or even

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impossible, due to the higher reduction potentials of the BNC cofactors (0.3-0.4 V for heme a3 [14, 22, 23] and 0.95 V for CuB [24]). This is supported by the experimental observation

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that the mitochondrial CcO is not able to reduce NO [25]. However, some CcOs are capable of NO-reduction, but the reason why this is and whether or not this is directly linked to the reduction potentials in the BNC remains to be determined (see section 4. Discussion). The largest CcO subfamily is the A-type, which is divided into two subgroups, A1 and A2, differing in the details of the proton donor in the main proton pathway, the D-pathway. The mitochondrial (bovine) enzyme and several other A1 CcOs from bacteria, have been shown not to reduce NO with a measurable rate (although slow NO-reduction activity was observed in the A1 quinol oxidase bo3 [26]), while at least one member of the A2 group, the Thermus (T.) thermophilus caa3 , has been shown to reduce NO at about 30 mol NO/mol caa3 x min [5]. At least one member of the B-type oxidases, the T. thermophilus ba3 , has also been shown to slowly reduce NO, about 3 mol NO/mol ba3 x min [5]. The fastest NO reduction in a CcO occurs in the C-type enzymes, labelled cbb3 , with about 100 mol

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NO/mol cbb3 x min [6, 7]. These values should be compared to the much faster 300-4500 mol NO/mol enzyme x min in ’true’ NORs [5]. The purpose of the present study is to elucidate the mechanism of NO reduction in CcO, compare it to the already characterised mechanism in cNOR and to compare the two in order to understand some general principles of what it takes to be an NO-reductase. For this, we use the same approach as in the previous studies on NO reduction in cNOR

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[19, 20], an approach that has also been used in several studies on O2 reduction in CcO (see e.g. [9]). Density functional theory (DFT) calculations are performed on models of

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the BNC, to construct free energy profiles for the entire catalytic cycles. The BNC model

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in previous studies on both cNOR and CcO mainly included the heme-group and the nonheme metal with ligands. The computational studies on CcO has been concerned with the A-type, which means that the BNC model consists of a heme a3 -Cu complex [9, 27], and

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a similar model is used here to study NO reduction to N2 O. The goal is to compare the mechanisms for NO reduction in cNOR and CcO, to further evaluate the conclusions drawn

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for cNOR, and also to use the obtained mechanisms to discuss why the NO reduction is so much slower or even impossible in CcO. An interesting observation is that the BNC as it is modelled here would be essentially identical regardless of whether it is based on X-ray

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structures for CcOs which do perform NO reduction (ba3 and caa3 , but not for cbb3 which has a different BNC heme) or those which do not perform NO reduction (aa3 ). This means

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that the model used in the present calculations should describe the chemistry occurring within the BNC in both these groups of CcOs, and that the differences observed in rates

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must come from properties outside of the active site.

2. Computational Details

The computational approach used here has been applied and tested for a large number of metallo-enzymes, as described in a recent review [28]. Quantum mechanical calculations are performed to investigate enzymatic reaction mechanisms in the following way. Geometric and electronic structures together with relative energies of different intermediates and transition states are determined for active site models using density functional theory (DFT). The relative energies are used to construct energy profiles for the entire catalytic cycles, and the heights of the rate-limiting barriers are compared to experimental rates when available, using transition state theory. To finally judge the likelihood of a particular

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reaction mechanism, the calculated energetics often has to be combined also with other types of experimental data, as will be described below. The calculations on NO reduction in CcO are performed on a model of the BNC, which mainly includes the heme a3 group and the CuB ion, together with all first shell amino acid ligands to the metals. All heme a3 substituents are kept in the model, except the long tail of the farnesyl group and the propionate groups. It was shown in a previous

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study on oxygen reduction in CcO that inclusion of the propionates and their immediate surrounding in the model did not change the computational picture of the reaction [9]. It

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should also be pointed out that charged groups in the surrounding of the active site have

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minor effects on the proton-coupled reduction potentials, since the charge effects are of similar size but with opposite signs on the electron and the proton uptakes. Also for the type of very short-distance electron transfer that occurs in the present reaction, from the

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metal ions to the substrate molecules located between the two metals ions, the net effects of charged groups in the surrounding are expected to be small. The tyrosine residue that

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is cross-linked to one of the histidine ligands on CuB , and which has been shown to be redox active in oxygen reduction is also included in the model, although it is not expected to be directly involved in the NO reduction. Furthermore, since the intermediates involved

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in NO reduction are more bulky than those involved in oxygen reduction, it was decided to include also the conserved valine near the BNC in the present model, see Fig. 2. The

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total number of atoms is about 160 (depending on the state) and the total charge of the model is +1, as obtained from a neutral heme-complex and a positively charged (+1) CuB

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complex. In the intermediates where only a proton or an electron is added, the charge is consequently changed to +2 or 0, respectively. The structure of the model is based on the X-ray coordinates for the Rhodobacter sphaeroides aa3 CcO [29], and it is noted that the BNC structures are close to identical in the different a3 -CuB CcOs discussed here. During geometry optimizations of the different intermediates a few atoms near the truncations are fixed to the X-ray coordinates to maintain some structural constraints from the surrounding protein. The fixed atoms are the alpha carbons, the hydrogen atoms replacing the peptide bonds plus one carbon atom on the porphyrin. This scheme to lock certain coordinates has been carefully tested in a large number of cases [30]. No water molecules are included in the model, apart from the one formed during the reduction process. The calculated energetics can therefore be considered as intrinsic reaction energies, which may be slightly modified by the presence of water molecules. The background to this choice is that the

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Figure 2: Models used for CcO (left) and cNOR (right) showing one NO bound in the BNC. Frozen atoms are marked with red circles. The most important spin-populations and distances (in ˚ A) are given.

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inclusion of just a few water molecules in the model, without a complete optimization of both the total number and the positions for each intermediate may distort the calculated energetics significantly (see also ref. [27]). As will be reported below the valine residue near

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the active site was found to have a significant effect on the energetics of NO reduction in CcO. Interestingly there is a valine present in the active site of cNOR in a similar position,

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and since an important purpose of the present study is to compare NO reduction in CcO and cNOR, new calculations on NO reduction in cNOR using a model that includes the valine were performed. Thus, the same procedure and model (based on the crystal structure from Pseudomonas (Ps) aeruginosa [31]) as in previous studies on NO reduction in cNOR ([18, 19]) is used, only the model is extended with the valine, see Fig. 2. Only the most important stationary points, i.e. those included in the presently reported energy profile are recalculated with the larger model. It has been found that the B3LYP-type of functionals in general give the best description of transition metal systems [28, 32], and in particular, by using the same type of functional in a large number of applications, many comparisons can be made, which means that its limitations will be known and can be taken care of, as will be described below. The

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dispersion corrected hybrid density functional B3LYP-D3 [33, 34] together with a double zeta basis with polarization functions on all second row atoms was used to optimize the geometries for the investigated intermediates and transition states. More accurate energies for the optimized structures were obtained using the B3LYP∗ -D3 [35] functional (with 15% exact exchange), the lacv3p+ basis for the metal ions [36] and the large cc-pvtz(-f) basis set for the rest of the atoms. Polarization effects on the relative energies from the omitted

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protein surrounding were estimated using a self-consistent reaction field approach with a dielectric constant of 4.0 [37]. Small dielectric effects were obtained for relative energies

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between intermediates with the same charge, and therefore these relative energies are not

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very sensitive to the choice of dielectric constant. Zero-point corrections were taken from the Hessians, calculated at the same level as the geometry optimizations, and using the harmonic approximation. The Jaguar 7.9 [36] program was used for all calculations except

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for the Hessians, for which the Gaussian 09 package [38] was used. Entropy effects were added to the relative energies only for the gaseous NO and N2 O molecules, and it is assumed

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that the entropy lost on binding or gained on release is equal to the translational entropy for the free molecule (10.8 kcal/mol for NO and 11.1 for N2 O at room temperature). For the binding enthalpy of a water molecule to bulk water an empirical value of 12 kcal/mol considered as free energies.

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is used, which includes explicit zero point effects. The energetic results reported below are

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To construct free energy profiles for the full catalytic cycle of NO reduction in a hemeCu oxidase, the energetics of the reduction steps has to be estimated. The energetics of

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one reduction step is obtained by comparing the reduction potential of the electron donor (in this case cytochrome c for both CcO and cNOR) with the proton-coupled reduction potential of the active site intermediate involved in the particular step. Since it is difficult to calculate reduction potentials and pKa values with the same absolute accuracy for systems in different surroundings, a procedure is used where the energies of the electrons and protons taken from outside the enzyme, i.e. 2H+ and 2e− in equation (1), are not calculated but estimated using experimental reduction potentials for the overall reaction, in combination with calculated values for the chemical part of the reaction, i.e. comparing the calculated energies for 2NO, N2 O and H2 O. This means that the sum of the energy of an electron from the donor and a proton from bulk water is fitted to a value that makes the calculations to reproduce the experimental overall energy of the catalytic cycle, in the same way as described in several previous studies [28]. Using the reduction potential 0.25

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V of the ultimate electron donor, cytochrome c, the overall reaction, i.e. the reduction of 2 NO to N2 O and water, with a potential of 1.177 V, becomes exergonic by 42.8 kcal/mol, taking into account that two electrons are needed. To reproduce this exergonicity at the present computational level requires a total energy of 383.8 kcal/mol for the electron from cytochrome c plus a proton from bulk water. This energy value can be partitioned into individual values for the electron and the proton using other experimental information,

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and following a previous study on NO reduction in cNOR the proton energy is set to 279.0 kcal/mol [19], giving a value of 104.8 kcal/mol for the electron from cytochrome c. With

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this partitioning of the energy also the individual steps of electron and proton transfer can

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be described, which, however, is not a main subject of the present study. A computational problem not taken care of by the above described procedure is the well-known fact that certain properties of heme-groups are not reproduced accurately by

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DFT, e.g. state-splittings [39, 40, 41, 42]. This means that the pure computational results for the heme-containing BNC in heme-copper oxidases have to be combined with some

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additional experimental information when the energy profiles are constructed, as described in previous computational studies [19, 27], and as will be shortly summarized here. First, from experiments it is known that certain intermediates in the CcO reaction with molecular

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oxygen have a high-spin coupling of the electrons on the heme-iron, and therefore, for the corresponding intermediates in the present study only high-spin heme a3 states are

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considered. Furthermore, it was found in earlier studies on both CcO and cNOR that the reduction potential of the ferric heme comes out significantly too small in the DFT

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calculations, and a correction on the order of 8-9 kcal/mol had to be introduced using similar types of BNC models and DFT-functional [9, 19]. Using the present model a correction of 9.4 kcal/mol should be used to reproduce the experimental value for the heme b3 reduction potential of 0.06 V for cNOR, very similar to the previously used correction of 9.2 kcal/mol for the slightly smaller model [19]. Using the same correction in the CcO reaction corresponds to a value of 0.39 V for the heme a3 reduction potential reasonably close to the experimental values V [14, 22], indicating that the same correction can be used for both systems. This means that the calculated heme Fe(III) proton-coupled reduction potential in the NO reduction process is increased by 9.4 kcal/mol in both the CcO reaction and the cNOR reaction. Clearly a corresponding (but opposite) correction has to be applied also on oxidation of the ferrous heme iron, i.e. when the hyponitrite is formed in the present reaction. Since it is expected that the main DFT-problem is concerned with the electronic

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Figure 3: Calculated free energy profile for one full catalytic cycle of NO reduction in cNOR. Starting from the reduced state, two NO molecules enter the active site and form a hyponitrite, from which the product N2 O molecule is released, resulting in the oxidized state with a µ-oxo-bridge. These chemical steps are followed by two proton-coupled reduction steps, regenerating the initial reduced state with a water molecule. part of the proton coupled reduction potential, the full correction of 9.4 kcal/mol is used

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for the hyponitrites in both CcO and cNOR. For CuB , on the other hand, the calculated reduction potential is not corrected, although it does not agree with the experimental value. The reason is that it has been argued in previous studies that the calculated value presumably better corresponds to the situation during catalytic turnover [24].

3. Results Below, the main results are presented, and evaluation and comparison between CcO and cNOR of the possible mechanisms in relation to experimental data will be made in section 4. Discussion.

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3.1 NO reduction in cNOR. To make a conclusive comparison between the NO reduction in CcO and cNOR, a new free energy profile for cNOR was constructed. The result is shown in Fig. 3, and the procedure used to construct this new energy profile differ in two ways from the most recently published energy profiles [18, 19]. First, to match the CcO calculations, an active

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site valine residue was added in the cNOR model used, which, however, turns out to have essentially no effect on the calculated energetics in cNOR. All stationary points shown in

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the energy profile in Fig. 3 are recalculated with the new model, and the relative energies obtained with the valine included differ from the ones obtained without the valine by less

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than one kcal/mol, which is in contrast to CcO as described below. Furthermore, in the present energy profile the energetics of the catalytic cycle is given relative to the ultimate

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electron donor cytochrome c, while in the previous publications the energetics were given relative to the immediate donor heme b [18, 19, 20]. This means that the entire reduction process described here is exergonic by 42.8 kcal/mol rather than 38.4 kcal/mol, a difference

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that mainly affects the two reduction steps. One main feature of the energy profile in Fig. 3 is that it describes a so called cis-b3 mechanism, and it was shown that the other suggested

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types of mechanisms (the trans-mechanism and the cis-FeB mechanism) are unlikely due to too high barriers [18, 43]. Another main feature of the mechanism described in Fig. 3 is that the chemical part of the reaction is essentially separated from the electron and proton

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uptake to the BNC. The first NO molecule reacts with the reduced BNC, removing the water molecule formed in the previous cycle. The second NO molecule enters and forms

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a hyponitrite, which after a rotation easily cleaves an N-O bond and releases the N2 O molecule, forming the experimentally observed oxidized intermediate with an oxo-bridge [44, 45]. The active site cofactors are then rereduced in two separate steps of protoncoupled electron transfer, leading to water formation in the BNC. These reduction steps are found to be rate-limiting for the entire catalytic reaction. This mechanism agrees with a large portion of experimental information [46, 47, 48] as discussed in previous computational papers [18, 43].

3.2 NO reduction in heme a3 -CcOs. The natural starting point for the study of NO reduction in CcO is to investigate if the same mechanism as in cNOR, labelled mechanism A, is possible also in CcO, and the 12

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Figure 4: Optimized structures for CcO: Reduced BNC with a bound water molecule (left), and the hyponitrite dianion (right) formed after the second NO enters. The most important spin-populations and distances (in ˚ A) are given. results will be described in subsection 3.2.1 below. In an early computational study a somewhat different mechanism was suggested for NO reduction in ba3 CcO, with one of

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the reduction steps occurring before the N-O bond cleavage [49], and such a mechanism, labelled mechanism B, is also investigated here, with the results presented in subsection

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3.2.2 below.

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3.2.1 Mechanism A for NO reduction in CcO. Since many experiments on the catalytic mechanism in the heme-copper oxidases traditionally have been performed starting with the fully reduced form of the enzyme, it is natural to start the description of the computational results with the reduced BNC. The first NO molecule that enters the reduced active site replaces the water molecule that was formed in the BNC in the previous cycle. The water molecule was found to be bound by 4.2 kcal/mol relative to the empty reduced BNC in the present model (Fig. 4), and the NO molecule was found to be bound by 14.4 kcal/mol relative to the empty BNC (Fig. 2). This means that the NO molecule is bound by 10.2 kcal/mol relative to the reduced H2 O-bound state of CcO, the starting point of the energy profile shown in Fig. 5. In agreement with experimental information [50] the most stable structure is formed with NO bound to the heme-iron (Fig. 2), and the calculations predict that there is only a weak minimum for NO 13

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Figure 5: Calculated free energy profile for one full catalytic cycle of NO reduction in CcO following mechanism A. Starting from the reduced state, two NO molecules enter the active site and form a hyponitrite, from which the product N2 O molecule is released, resulting in the oxidized state with a µ-oxo-bridge. These chemical steps are followed by two protoncoupled reduction steps, regenerating the initial reduced state with a water molecule. The grey curve shows the computational results obtained with a smaller model without the Val287. The dashed curves indicate that these barriers are not studied here. bound to CuB , in fact, of higher energy than the water molecule bound to the BNC. The first chemical step in the catalytic cycle is the formation of the N-N bond after a second NO molecule has entered the BNC, and it is first investigated if two NO molecules can bind simultanously in the BNC. Many different types of structures were tried to investigate if the second molecule could bind to CuB , or at all bind in the center of the BNC, at the same time as the first NO binds to the heme-iron. The lowest energy was obtained for a structure where the second NO does not really coordinate to CuB (Cu-N distance of ˚ but the energy surface is quite flat, which means that it is difficult to determine 3.5 A), an exact structure for the BNC complex with two NO molecules. However, all structures of this kind were found to be high in energy, which means that the second NO molecule is unbound by 9-10 kcal/mol relative to the structure with one NO bound to heme a3 and

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one NO molecule in the gas phase, when the loss of entropy is taken into account, which is here set to 10.8 kcal/mol at room temperature. However, this also means that at very low temperatures, where there is essentially no entropy gain in releasing the weakly bound NO molecule, it can still be bound by a few kcal/mol, in agreement with experimental observations at low temperature of BNC complexes with two NO molecules bound [51]. Although the second NO molecule is not really bound in the BNC, it was found that

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an N-N bond can be formed with a rather low energy barrier, with the nitrogen of the second NO attacking the first one still bound to heme a3 . In the same way as in cNOR a

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hyponitrite intermediate is formed with a low barrier, here calculated to be approximately

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12 kcal/mol relative to the intermediate with only one NO molecule bound in the BNC (and +1.8 kcal/mol relative to the zero-point of the energy diagram in Fig 5), which is only slightly higher than the entropy loss. In spite of the low barrier, the process for the N-N

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bond formation is rather complicated, which means that the height of the barrier is rather uncertain. During the first part of the process there is mainly a coupling occurring between

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the two unpaired electrons, one on each NO molecule, and the energy surface is quite flat, ˚ At shorter N-N distances, an electron at least for N-N distances between 2.6 and 2.1 A.

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transfer occurs from the heme a3 iron, resulting in a hyponitrite radical with Fe(III) and ˚ During this process the calculated energy decreases Cu(I) and an N-N distance of 1.44 A. slightly, but as discussed in the computation details the calculated reduction potential of

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the heme iron is too low, and a correction must be introduced, which implies that also this part of the energy surface is quite flat. A structure with a hyponitrite radical has a similar

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energy as in the initial part of the N-N bond formation, when corrected for the error in the heme reduction potential. The hyponitrite radical is not very stable, and only a small perturbation of the structure finally leads to the transfer of another electron, this time from CuB , and results in formation of the hyponitrite dianion intermediate with Fe(III) and Cu(II). The geometric and electronic structure of the hyponitrite intermediate formed after this rather complicated bond-forming step is shown in Fig. 4, and the energetics of this reaction step is described in Fig. 5, showing that the energy of the dianionic hyponitrite intermediate is estimated to be at -6.3 kcal/mol relative to the reduced starting point, while the nitrosyl intermediate is at -10.2 kcal/mol. At this point an important difference between cNOR and CcO can be seen, and that is the difference in exergonicity of this reaction step. In cNOR the formation of the hyponitrite is quite exergonic, by 6.8 kcal/mol, while in CcO it appears to be endergonic by 3.9 kcal/mol. The source of this difference will be

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analyzed in section 4. Discussion below. It should be recalled that for this part of the energy profiles; hyponitrite formation, quite large corrections are introduced, making the given values uncertain, but since the same correction is used in both CcO and cNOR, the relative difference should be more reliable. The second chemical step in the formation of the N2 O product molecule is to cleave one of the N-O bonds in the hyponitrite intermediate. It was found for cNOR that the

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hyponitrite had to be rotated into a structure with one of the oxygen atoms bridging between the two metals before this could occur with a low barrier, see Fig. 3. This is true

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also for the reaction in CcO, since just increasing the N-O bond distance in the original

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hyponitrite intermediate leads to very high energies. A rotated hyponitrite can be formed also in CcO, see Fig. 6. Again there is a significant difference in the energetics between the two enzymes. In cNOR the barrier for the rotation is low, only 11.9 kcal/mol, the energy

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of the rotated hyponitrite is rather similar to the original hyponitrite structure, and finally the barrier for cleavage of the N-O bond is low, only 6.6 kcal/mol relative to the rotated

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hyponitrite, see Fig. 3. In CcO on the other hand, the rotated hyponitrite structure is 6.8 kcal/mol above the original hyponitrite structure, and the intrinsic barriers are high, 19.5 kcal/mol for rotation of the hyponitrite, and 11.5 kcal/mol for cleaving the N-O bond,

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relative to the already high energy rotated hyponitrite, leading to a total barrier of 23.4 kcal/mol, see Fig. 5 and the discussion below.

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Finally, when the N2 O molecule leaves, the BNC is left in its oxidized form, CuB (II) and Fea3 (III) with a bridging oxo-group, see Fig. 6, similar to the oxidized state of cNOR,

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see Fig. 3. This reaction step is exergonic by 18.2 kcal/mol relative to the initial, most stable hyponitrite intermediate, including the entropy gain of 11.1 kcal/mol by the free N2 O molecule. The corresponding exergonicity in cNOR is 27.0 kcal/mol. It is noted that the calculations give a somewhat lower energy (3.0 kcal/mol) for a ferryl-oxo-Cu(I) structure of the oxidized state, but to simplify the comparison to cNOR only the Fe(III)-µ-oxo-Cu(II) bridged structure is reported in the energy profiles and in the discussion. The remaining part of the catalytic cycle, after the N2 O molecule has left, corresponds to two reduction steps, taking up a total of two electrons and two protons, forming the reduced form of the BNC with one newly formed water molecule, see Fig. 4. In the first proton-coupled reduction step CuB is reduced and a bridging hydroxide is formed, see Fig. 7, and this reduction step is found to be exergonic by 15.0 kcal/mol, see Fig. 5. The Fea3 is reduced in the second reduction step, with an exergonicity of 3.3 kcal/mol, a

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Figure 6: Optimized structures for CcO: Rotated hyponitrite (left) in the BNC, and the µ-oxo intermediate (right) formed after N2 O has left the BNC. The most important spinpopulations and distances (in ˚ A) are given. value that is obtained by adding a correction to the calculated value, as described in the section 2. Computational Details. The reduction steps are not studied in detail here, but

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the calculations show that for both steps, it is energetically most favorable to transfer the proton to the BNC before the electron, rather than the opposite order. The barriers are

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assumed to be determined by the proton transfer processes, and they can not be determined using the present computational procedure.

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An interesting result concerns the role of the conserved valine (Val-278, R. sphaeroides aa3 numbering) residue in the active site of CcO. This valine is conserved across the hemecopper oxidase superfamily (i.e. also to C-type and to NOR) and was shown by site-directed mutagenesis to influence the delivery of substrate O2 in A and B CcOs [52, 53, 54]. Although conserved, the valine could play different roles in different CcOs, as it forms part of different conserved motifs in the different subfamilies, e.g. in A1 CcO, it is the V in the HPEVY motif, where the E is the proton donor Glu-286 (R. sphaeroides numbering), missing in all other families, and the Y is the active site Tyr-288, which is absent in NORs and Ctype CcOs (but in C-type CcOs an equivalent tyrosine is present at a different location [55]). As described in the Computational details, the current CcO model includes this Val-278 near the BNC, and the calculations indicate that it has a significant effect on the energetics for Mechanism A. Thus, results obtained using a model without the valine is

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Figure 7: Optimized structures for CcO: Reduced (and protonated) hyponitrite (left) in the BNC, and the bridging hydroxyl intermediate (right) formed after N2 O has left the BNC.The most important spin-populations and distances (in ˚ A) are given. shown as the light grey profile in Fig. 5 for comparison. The valine has essentially no effect on the initial and final parts of the reaction, and therefore only the part where the

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energetics differs significantly between the two models are given in the figure. Accordingly, the initial hyponitrite intermediate is very close in energy for the two models, differing

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by less than one kcal/mol. However, the high energy (+0.5 kcal/mol) obtained for the rotated hyponitrite using the large model is an effect of the presence of the valine residue,

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since without the valine the rotated hyponitrite is at about the same level as the initial hyponitrite intermediate (-7.0 kcal/mol), which is similar to the energy profile for cNOR shown in Fig. 3, where the two hyponitrites are at -19.1 and -17.7 kcal/mol, respectively. Thus, there is a repulsive effect from the valine when the hyponitrite is rotating in the BNC of CcO, which increases the energy by 4.3 in TSrot , by 7.5 kcal/mol in the intermediate with the rotated hyponitrite and by 7.0 kcal/mol in TSN −O . These explicit values may change somewhat if a larger more flexible model could be used, but it is quite unlikely that the effect should disappear. 3.2.2 Mechanism B for NO reduction in CcO. Starting from the reduced BNC with a bound water molecule, the second mechanism investigated is identical to the mechanism A until the formation of the hyponitrite inter-

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Figure 8: Calculated free energy profile for one full catalytic cycle of NO reduction in CcO following mechanism B. Starting from the reduced state, two NO molecules enter the active site and form a hyponitrite. After a proton-coupled reduction of the hyponitrite the product N2 O molecule is released, resulting in the state with a µ-hydroxo-bridge. These chemical steps are followed by one proton-coupled reduction step, regenerating the initial reduced state with a water molecule.The dashed curves indicate that these barriers are not studied here.

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mediate (Fig. 4). The calculations show that it is thermodynamically favorable to transfer an electron and a proton to the BNC already at this point, i.e. to reduce the hyponitrite intermediate forming the intermediate with a protonated hyponitrite shown in Fig. 7. The calculations indicate that the overall energetics of such a reduction step is slightly exergonic, see Fig. 8, although it should be recalled that the energy of the initial hyponitrite is uncertain due to the large correction. The barrier is most likely determined by proton transfer into the BNC, which is not possible to determine using the present model, or type of computational approach. The calculations indicate that this reduction step can either be initiated by electron or by proton transfer to the BNC. The calculated electron affinity of the hyponitrite intermediate is only 7 kcal/mol lower than the value used for the electron donor, and the calculated proton affinity is 17 kcal/mol lower than the value used for the proton donor. Interestingly it is the heme iron that becomes reduced in this intermediate, 19

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see Fig. 7, which is in agreement with previous results [49], the corresponding intermediate with CuB reduced is a few kcal/mol higher in energy for this type of structure. This result is in contrast to the two reduction steps in the rereduction process discussed above for Mechanism A, where CuB is reduced before the heme iron. Obviously the reduction potential of the two metal complexes varies with the chemical surrounding, i.e. in this case with the central ligand. Finally, since the mechanism for oxygen reduction in CcO involves

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the creation of a neutral tyrosyl radical by the transfer of an electron and a proton from the active site tyrosine in the O-O bond cleavage step [9], it was also tried to use an electron

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and a proton from the tyrosine for the reduction of the center of the BNC in the hyponitrite

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intermediate, rather than taking them from outside the enzyme. However, such a reaction step was found to be significantly endergonic (by about 16 kcal/mol), and does therefore not seem likely.

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In the one-electron reduced state with a protonated hyponitrite the barrier for cleaving the N-O bond that leaves a hydroxyl group between the metals in the BNC is quite low, only

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4.3 kcal/mol relative to the reduced hyponitrite, see Fig. 8. In the intermediate obtained after the N2 O molecule has left, CuB is reduced and the heme-iron is oxidized, see Fig. 7, in accordance with the corresponding intermediate in Mechanism A. This reaction step is

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exergonic by 30.7 kcal/mol relative to the one-electron reduced hyponitrite, including the entropy gain of 11.1 kcal/mol from releasing the N2 O molecule. Since one reduction step

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has already occurred, there is only one reduction step left to complete the catalytic cycle after the N2 O molecule was formed. This reduction step is identical to the last step in

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Mechanism A. Finally, for the structures involved in this mechanism, the conserved valine has no energetic effects.

4. Discussion

4.1 Comparison between NO reduction in cNOR and heme a3 -CcO. To simplify the comparisons between NO reduction in cNOR and CcO, the two energy profiles for the same type of mechanism (Mechanism A) obtained from the calculations are shown together in Fig. 9. Since the ultimate electron donor is the same (cytochrome c) the overall exergonicity is the same for the two reactions, but a closer comparison of the two energy profiles elucidates some important differences that can be summarized as follows. In cNOR there is a rather strong driving force for the chemical steps in which the two NO 20

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Figure 9: Comparison between the calculated free energy profiles for NO reduction in CcO, mechanism A (green curve, same as Fig. 5), and cNOR (brown curve, same as Fig. 3). The dashed curves indicate that these barriers are not studied here. molecules are reduced into N2 O by the reduced BNC, while the rereduction of the BNC

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cofactors is endergonic. In contrast, in CcO there is a significantly smaller driving force for the chemical steps of N2 O formation, in fact some steps are even endergonic, while the

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rereduction of the BNC cofactors is significantly exergonic. This is due to the differences in the reduction potentials of the active sites such that there is a natural “trade-off” when these potentials are raised in CcOs as compared to cNOR, i.e. the rapid initial electron transfer to NO using the electrons already present in the active site becomes slower whereas the rereduction becomes faster. Comparing the two energy profiles in Figure 9, the effects of the differences in reduction potentials of the BNC cofactors can be described more in detail. First, the differences in the proton-coupled BNC reduction potentials appear explicitly in the two rereduction steps, i.e. the later part of the energy profiles. In the very last reduction step the heme iron is reduced from ferric to ferrous, and in CcO this step is exergonic by 3.3 kcal/mol, while in cNOR it is endergonic by 4.4 kcal/mol, a difference of approximately 0.3 V (7.7 kcal/mol),

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as obtained from the experimentally observed proton-coupled reduction potentials, 0.3-0.4 V for heme a3 in bovine CcO [14, 22, 23] and 0.06 V for heme b3 in cNOR [21], respectively. For the first reduction step, i.e. the second last step in the energy profiles, the difference in exergonicity is about 0.6 V (13.9(15.0-1.1) kcal/mol), in favor of CcO. This difference is obtained from the difference between the previously established computational result for the proton coupled reduction potential of CuB (II) of about 0.95 V [24] and the experimental

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value for FeB (III) of 0.32 V [21].

Second, since the two energy profiles in Figure 9 have the same starting- and end-points

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the difference in the BNC reduction potentials between CcO and cNOR obviously must

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affect the chemical part of the reaction in the same (but opposite) way as the reduction steps discussed above. The same difference of 21.6 kcal/mol (7.7+13.9 kcal/mol) is obtained for the reaction energy from the fully reduced reactant to the oxo-bridged intermediate

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formed when the N2 O molecule has left, this time with the cNOR reaction being the most exergonic, 46.1 kcal/mol in cNOR as compared to 24.5 kcal/mol in CcO. The energy profiles

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in Figure 9 also show that the larger driving force for the chemical steps in cNOR also leads to lower barriers for this part of the catalytic cycle, as compared to CcO. The rate limiting barrier for the chemical part in cNOR is only 11.9 kcal/mol, and this is not the rate limiting

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step for the entire catalytic cycle. The corresponding step in the CcO reaction has a total barrier of 23.4 kcal/mol, which makes the disappearance of NO much slower.

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In summary, the above discussed results support the previous suggestion that the role of the low cNOR potentials is to optimize the rate of scavenging of the toxic NO molecule.

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The results also show that the price to be paid for the fast NO disappearance is a slow rereduction of the low-potential BNC. That this energetically unfavorable rereduction process is already slow was also suggested to explain why it is not coupled to electrogenic proton transfer. The lack of electrogenicity in turn explains the lack of free energy storage in this overall exergonic reduction reaction [19, 20]. In contrast, when CcO reacts with its natural substrate, molecular oxygen, the single chemical step, the cleavage of the O-O bond, can occur with a low barrier of 12.4 kcal/mol [56], at least partly due to involvement of the active site tyrosine [57], and at the same time without wasting too much energy. The exergonicity for the reaction from the reduced water-bound BNC to the intermediate with the O-O bond cleaved (PM ), formed in the single, four electron reduction step after O2 binding, is found be about 6 kcal/mol [9]. Thus, due to the high reduction potentials in the CcO BNC, the rest of the catalytic cycle

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consists of four significantly exergonic reduction steps, which can all contribute to energy storage through electrogenicity and proton pumping. A more detailed comparison between the energy diagrams for NO reduction in CcO and cNOR allows some further conclusions. First, the oxidation of the two BNC metals occurs already upon formation of the first dianionic hyponitrite intermediate, and the difference between the two enzymes in exergonicity relative to the reduced starting point of the

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energy diagrams is only 12.8 (19.1-6.3) kcal/mol, as compared to a difference in reduction potentials of 21.6 kcal/mol discussed in the previous paragraph. The larger difference in

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reduction potentials refers to the oxo- and hydroxo-bridged intermediates, which are the

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relevant complexes for the reductive part of the cycle. These results are in accordance with the well-known fact that the reduction potentials of metal complexes depend strongly on the chemical environment of the metal atoms, in this case the nature of the bridging ligand.

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Another more specific conclusion concerns the role of a conserved valine residue in the active sites of both CcO and cNOR. As described above this valine turns out to have a

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significant effect on the energetics for Mechanism A in CcO, but no effect on the energetics in cNOR. This difference between the two enzymes is probably explained by minor differences in the location of the valine, in particular, in CcO both side-chain methyl groups point

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towards the center of the BNC, while in cNOR only one of the methyl groups points towards the center. A comparison between the energy profile for cNOR in Fig. 3 and the

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energy profile for CcO using a model without the valine, grey curve in Fig. 5, confirms the conclusions above that it is mainly the higher reduction potentials of the BNC metals in

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CcO that leads to a lower driving force and also to higher barriers for the NO reduction process. Thus, already without the valine in the CcO model, the calculated rate-limiting barrier for the chemical part of the reaction is raised from 11.9 kcal/mol in cNOR to 19.1 kcal/mol in CcO. However, as can be seen in Fig. 5 the barrier increases further to 23.4 kcal/mol when the valine is included. It can therefore be concluded that the active site valine (Val287) seems to play an important role in preventing a too fast reduction of NO in CcO, at least via Mechanism A, while the corresponding active site valine in cNOR, Val210 (next to the Glu211 coordinating to FeB ) has no effect. Whether or not the valine plays such a role in the physiological context is not clear, but we note that NO is an important reversible regulator of mitochondrial CcO activity (reviewed in [58]), such that it could be important to tightly control what does and does not happen upon NO binding.

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4.2 Comparisons to experimental results for NO reduction in different HCuOs. As described in the introduction, some CcOs can reduce NO to N2 O, but at a slower rate than cNOR. The barrier corresponding to the overall rate of NO reduction in cNOR is 15-16 kcal/mol [19, 46], with the rereduction as the rate limiting part of the catalytic cycle. As mentioned above the calculated rate limiting barrier for the chemical part of the

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reaction in cNOR is only 11.9 kcal/mol. The barriers corresponding to the experimental overall rates of NO reduction in those CcOs with a heme a3 -CuB active site that actually

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perform this reaction at a measurable rate should be 18-19 kcal/mol (3-30 mol NO/mol enzyme x min) [5], but it is not known from experiment which part of the reaction is rate

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limiting. It can also be estimated from results in ref. [5] that the rate-limiting barrier for bovine aa3 CcO, which does not perform NO reduction at an observable rate, is at least 21

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kcal/mol. Thus, the range of barriers for a3 -CuB oxidases, 18-21 kcal/mol, is not very large from a computational point of view, meaning that the differences between the enzymes are within the uncertainty of the calculations, which makes it difficult to explain the source of

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the differences between the different species, in particular considering the large corrections that has to be applied to the calculated values. However, some conclusions about the

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most likely reaction mechanism for NO reduction in CcO should be possible to draw when the combination of the experimental and computational results are considered, as will be discussed below.

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An interesting experimental result from ref. [5] for the bovine enzyme (aa3 ), which is one of the CcOs that does not reduce NO, is that one NO molecule binds to the reduced BNC,

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with no further reaction. In the same study it was found that also for the ba3 CcO, one for which NO reduction is actually observed, the same intermediate with only one NO molecule bound in the BNC was observable [5]. The calculated barrier for hyponitrite formation, 12 kcal/mol, is too low to prevent a fast formation of the hyponitrite, which is true even if it would be underestimated by a few kcal/mol, see Fig. 5. Therefore, the explanation for the experimental observation of only the nitrosyl intermediate has to be that the hyponitrite is several kcal/mol less stable than the nitrosyl intermediate, making its concentration too low to be observable. As described in the section 2. Computational Details, a large correction of 9.4 kcal/mol was applied for the position of the hyponitrite, due to an error in the calculated reduction potential of the ferric heme. With this correction the hyponitrite ends up 3.9 kcal/mol above the nitrosyl intermediate (Fig. 5), in good agreement with the experimental observations [5], which therefore can be taken to support the correction made 24

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to the computational results. Together with this experimental observation, the calculations indicate that the rate limiting barrier for the chemical part of the reaction for Mechanism A is 23.4 kcal/mol, corresponding to the TSrot transition state in Fig. 5. This high barrier indicate that the NO reduction reaction in CcO does not occur via Mechanism A, even if it can not be completely excluded, due to the uncertainty in the calculated relative energies. On the other hand, there are also experimental results interpreted to show a hyponi-

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trite intermediate during the reaction between NO and CcO, in both ba3 [59] and caa3 [60]. These observations may indicate that the energy profile for the CcO reaction in Fig. 5 is

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not completely correct. To make the hyponitrite observable its energy must be significantly lower, much closer to the nitrosyl intermediate. Interestingly the experimentally reported

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N-N frequencies for the hyponitrite intermediate, 1334 cm−1 [59] and 1338 cm−1 [60], respectively, agree reasonably well with the harmonic frequency of 1398 cm−1 obtained from

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the present Hessian calculations for the hyponitrite intermediate. Furthermore, a lower energy for the hyponitrite intermediate, should probably also lower the barrier for the

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chemical part of the energy profile, giving a value closer to the rates observed for the heme a3 copper oxidases, making mechanism A more likely. However, an important aspect of mechanism A is that the chemical part of the energy profile depends only on the chemistry

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occurring in the center of the BNC, and it will therefore be more or less identical for all CcOs with a heme a3 -CuB active site. This means that with this kind of mechanism the

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discrimination between CcOs that do perform NO reduction at a measurable rate and those that do not would have to occur in the reduction steps following after N2 O has formed.

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This interpretation is contradicted by the fact that the bovine enzyme stops already at the nitrosyl intermediate. It is therefore concluded that Mechanism A most likely should be considered as not compatible with experimental observations for NO reduction in CcOs. In contrast to Mechanism A, the alternative mechanism investigated, Mechanism B, actually presents a possibility to explain why different CcOs with the same heme a3 -CuB active site behaves differently with respect to NO reduction. The free energy profile for Mechanism B is given in Fig. 8. The first two steps, forming the nitrosyl and the dianionic hyponitrite intermediates are the same as for Mechanism A, which means that the position of the hyponitrite depends on the applied correction as discussed above. The calculations show that it is thermodynamically favorable to take up an electron and a proton to the dianionic hyponitrite intermediate, forming a protonated monoanionic hyponitrite, which is only about one kcal/mol above the nitrosyl intermediate. From this point the N-O bond can

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be easily cleaved, with a barrier of only 4.3 kcal/mol relative to the protonated hyponitrite. What is most interesting with this mechanism is that the barrier for the proton-coupled electron transfer to the dianionic hyponitrite intermediate is most likely rate limiting for the N2 O formation, and that the barrier for this reaction step may very well be different in different CcOs with the same BNC, since the different types of CcOs have quite different proton channels. The A-type CcO has two proton channels, the D- and the K-channel, and

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at the corresponding stage of oxygen reduction the D-channel is known to be used. It is also known that the A1- and A2- D-channels have essential differences. For B-type CcO there

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is only one proton channel, referred to as the K-analogue channel. Thus, to fit with the

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experimental data, for the A1(aa3 )-type of CcO the total barrier for this proton-coupled reduction step should be at least 21 kcal/mol to explain why the reaction stops at the nitrosyl formation. For the A2(caa3 )- and the B(ba3 )-families of CcO on the other hand,

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the total barriers for this step should be at most 18-19 kcal/mol, since it is not known if this step or the last proton-coupled reduction step is the overall rate limiting step in the

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catalytic cycle.

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5. Conclusions and Suggestions for Further Experimental Studies The results from quantum chemical calculations, in combination with available experi-

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mental information indicate that NO reduction in CcOs with a heme a3 -CuB active site most likely does not occur with the same mechanism as NO reduction in cNOR, in which N2 O

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is formed using only electrons present in the reduced BNC. The reason is that the higher reduction potentials in CcO together with an active site valine create too high barriers for such a reaction mechanism. On the other hand, the calculation indicate that it should be thermodynamically feasible to reduce the hyponitrite intermediate in a proton-coupled step followed by a fast N-O bond cleavage. The barrier for such a proton-coupled reduction step may very well be different in different CcO, which can explain why different CcO with the same a3 -CuB active site behave different with respect to NO reduction. It would therefore be very interesting to investigate experimentally whether or not it is possible to form N2 O in ba3 or caa3 CcOs with only the active site reduced. Furthermore, the properties of the populated intermediates during turnover as well as details on the kinetics of single-turnover NO-reduction in the CcOs would help shed further light on this mechanism in the future.

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ASSOCIATED CONTENT Supporting Information Available: Contains cartesian coordinates for the most important structures, i.e. the structures shown in the figures in the main text.

Funding

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This work was supported by the Swedish Research Council (grant number 2016-03721).

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Computer time was provided by the Swedish National Infrastructure for Computing.

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References

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[1] Fujiwara, T., Fukumori, Y. (1996) Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans Atcc 35512, J. Bacteriol.

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178, 1866-1871.

[2] Butland, G., Spiro, S., Watmough, N. J., and Richardson, D. J. (2001) Two conserved glutamates in the bacterial nitric oxide reductase are essential for activity but not

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assembly of the enzyme, J. Bacteriol. 183, 189-199. ¨ [3] Flock, U., Watmough, N.J., Adelroth, P. (2005) Electron/Proton Coupling in Bacterial

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Nitric Oxide Reductase during Reduction of Oxygen, Biochemistry 44, 10711-10719. ¨ [4] Blomberg, M.R.A., Adelroth, P. (2017) The mechanism for oxygen reduction in cy-

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tochrome c dependent nitric oxide reductase (cNOR) as obtained from a combination of theoretical and experimental results, Biochim. Biophys. Acta 1858, 884-894. [5] Giuffr`e, A., Stubauer, G., Sarti, P., Brunori, M., Zumft, W.G., Buse, G., Soulimane, T. (1999) The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: Evolutionary implications, Proc. Natl. Acad. Sci. USA 96, 14718-14723. [6] Forte, E., Urbani, A., Saraste, M., Sarti, P., Brunori, M., and Giuffr`e, A. (2001) The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity, Eur. J. Biochem. 268, 6486-6491. ¨ [7] Huang, Y., Reimann, J., Lepp, H., Drici, N., and Adelroth, P. (2008) Vectorial proton transfer coupled to reduction of O2 and NO by a heme-copper oxidase, Proc. Natl. Acad. Sci. USA 105, 20257-20262. 27

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[8] Pereira, M.M., Santana, M., Teixeira, M. (2001) A novel scenario for the evolution of heme-copper oxygen reductases, Biochim. Biophys. Acta 1505, 185-208. [9] Blomberg, M.R.A. (2016) The mechanism of oxygen reduction in Cytochrome c oxidase and the role of the active site tyrosine, Biochemistry 55, 489-500. ¨ [10] Lee, H.J., Reimann, J., Huang, Y., and Adelroth, P. (2012) Functional proton transfer

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pathways in the heme-copper oxidase superfamily, Biochim. Biophys. Acta 1817, 537-

RI

544.

[11] Chang, H.Y., Hemp, J., Chen, Y., Fee, J.A., Gennis, R.B. (2009) The cytochrome

SC

ba(3) oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping Proc. Natl. Acad. Sci. USA

NU

106, 16169-16173.

[12] Hemp, J., Han, H., Roh, J.H., Kaplan, S., Martinez, T.J., Gennis, R.B. (2007) Com-

MA

parative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb(3) oxidase) of heme-copper oxygen reductases Biochemistry 46, 9963-9972.

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[13] Brzezinski, P., Gennis, R.B. (2008) Cytochrome c oxidase: exciting progress and re-

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maining mysteries, J. Bioenerg. Biomembr. 40, 521-531. [14] Kaila, V.R.I., Verkhovsky, M.I., Wikstr¨om, M. (2010) Proton-Coupled Electron Trans-

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fer in Cytochrome Oxidase, Chem. Rev. 110, 7062-7081. [15] Popovic, D. M., Leontyev, I.V., Beech, D.G., Stuchebrukhov, A.A. (2010) Similarity of cytochrome c oxidases in different organisms, Proteins 78, 2691-2698. [16] Blomberg, M.R.A., Siegbahn, P.E.M. (2014) Proton pumping in Cytochrome c oxidase: energetic requirements and the role of two proton channels, Biochim. Biophys. Acta. 1837, 1165-1177. [17] Rich, P.R. (2017) Mitochondrial cytochrome c oxidase: catalysis, coupling and controversies Biochem. Soc. Transactions 45, 813-829. [18] Blomberg, M.R.A. (2017) Can reduction of NO to N2 O in cytochrome c dependent nitric oxide reductase (cNOR) proceed through a trans-mechanism? Biochemistry 56, 120-131. 28

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[19] Blomberg, M.R.A., Siegbahn, P.E.M. (2016) Improved free energy profile for reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR) J. Comput. Chem. 37, 1810-1818. [20] Blomberg, M.R.A., Siegbahn, P.E.M. (2013) Why is the reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR) not electrogenic? Biochim. Biophys. 1827,

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826-833. [21] Gr¨onberg, K.L.C., Roldan, M.D., Prior, L., Butland, G., Cheessman, M.R., Richard-

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son, D.J., Spiro, S., Thomson, A.J., Watmough, N.J. (1999) A low redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the

SC

evolution of energy-conserving heme-copper oxidases, Biochemistry 38, 13780-13786.

in cell respiration, Nature 356, 301-309.

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[22] Babcock, G.T., Wikstr¨om, M. (1992) Oxygen activation and the conservation of energy

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[23] Jancura, D., Berka, V., Antalik, M., Bagelova, J., Gennis, R.B., Palmer, G., Fabian, M. (2006) Spectral and kinetic equivalence of oxidized cytochrome c oxidase as isolated and “activated” by reoxidation, J Biol Chem 281, 30319-30325.

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[24] Blomberg, M.R.A., Siegbahn, P.E.M. (2015) Protonation of the binuclear active site in cytochrome c oxidase decreases the reduction potential of CuB , Biochim. Biophys.

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Acta. 1847, 1173-1180.

[25] Stubauer, G., Giuffr`e, A., Brunori, M., Sarti, P. (1998) Cytochrome c Oxidase Does

AC CE

Not Catalyze the Anaerobic Reduction of NO, Biochem. Biophys. Res. Comm. 245, 459-465.

[26] Butler, C.S., Forte, E., Scandurra, F.M., Arese, M., Giuffre, A., Greenwood, C., Sarti, P. (2002) Cytochrome bo3 from Escherichia coli: the binding and turnover of nitric oxide, Biochemical and Biophysical Research Communications 296, 1272-1278. [27] Blomberg, M.R.A., Siegbahn, P.E.M. (2015) How cytochrome c oxidase can pump four protons per oxygen molecule at high electrochemical gradient Biochim. Biophys. Acta. 1847, 364-376. [28] Blomberg, M.R.A., Borowski, T., Himo, F., Liao, R.-Z., Siegbahn, P.E.M. (2014) Quantum Chemical Studies of Mechanisms for Metalloenzymes, Chem. Rev. 114 36013658. 29

ACCEPTED MANUSCRIPT

[29] Qin, L., Hiser, C., Mulichak, A., Gavarito, R.M., Ferguson-Miller, S. (2006) Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase, Proc. Natl. Acad. Sci. USA 103, 16117-16122. [30] Siegbahn, P.E.M. and Borowski, T. (2006) Modeling Enzymatic Reactions Involving Transition Metals, Acc. Chem. Res. 39, 729-738.

PT

[31] Hino, T., Matsumoto, Y., Nagano, S., Sugimoto, H., Fukumori, Y., Murata, T., Iwata, S., Shiro, Y. (2010) Structural Basis of Biological N2 O Generation by Bacterial Nitric

RI

Oxide Reductase, Science 330, 1666-1670.

SC

[32] Siegbahn, P.E.M. and Blomberg, M.R.A. (2014) Energy Diagrams for Water Oxidation in Photosystem II Using Different Density Functionals, J. Chem. Theory Comput. 10,

NU

268-272.

[33] Becke, A.D. (1993) Density-functional thermochemistry. III. The role of exact ex-

MA

change, J. Chem. Phys. 98, 5648–5652.

[34] S. Grimme, J. Anthony, S. Ehrlich, H. Krieg (2010) A consistent and accurate ab

ED

initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu J. Chem. Phys. 132, 154104.

PT

[35] Reiher, M., Salomon, O. and Hess, B. A. (2001) Reparameterization of hybrid functionals based on energy differences of states of different multiplicity, Theor. Chem.

AC CE

Acc. 107, 48–55

[36] Jaguar 7.6 (2009) Schr¨odinger, LLC, New York, NY. [37] Blomberg, M.R.A., Siegbahn, P.E.M. and Babcock, G.T. (1998) Modeling electron transfer in biochemistry: A quantum chemical study of charge separation in Rhodobacter sphaeroides and photosystem II, J. Am. Chem. Soc.120, 8812-8824. [38] Gaussian 09, 2010 Revision C.01, Gaussian Inc., Wallingford, CT. [39] Radon, M., Pierloot, K. (2008) Binding of CO, NO, and O2 to Heme by Density Functional and Mulitireference ab Initio Calculations, J. Phys. Chem. A 112, 1182411832.

30

ACCEPTED MANUSCRIPT

[40] Vancoillie, S., Zhao, H., Radon, M., Pierloot, K. (2010) Performance of CASPT2 and DFT for Relative Spin-State Energetics of Heme Models, J. Chem. Theory Comput. 6, 576-582. [41] Strickland, N., Harvey, J.N. (2007) Spin-Forbidden Ligand Binding to the FerrousHeme Group: Ab Initio and DFT Studies, J. Phys. Chem. B 111, 841-852.

RI

Initio Calculations, J. Phys. Chem. A 113, 7338-7345.

PT

[42] Olah, J., Harvey, J.N. (2009) NO bonding to Heme Groups: DFT and Correlated av

[43] Blomberg, M.R.A., Siegbahn, P.E.M. (2012) Mechanism for N2 O Generation in Bacte-

SC

rial Nitric Oxide Reductase: A Quantum Chemical Study, Biochemistry 51, 5173-5186. [44] Mo¨enne-Loccoz, P., de Vries, S. (1998) Structural Characterization of the Catalytic

NU

High-Spin Heme b of Nitric Oxide Reductase: A Resonance Raman Study, J. Am. Chem. Soc. 120, 5147-5152.

MA

[45] Mo¨enne-Loccoz, P., Richter, O.-M.H., Huang, H. Wasser, I.M. Ghiladi, R.A., Karlin, K.D., de Vries, S. (2000) Nitric Oxide Reductase from Paracoccus denitrificans

ED

Contains an Oxo-Bridged Heme/Non-Heme Diiron Center, J. Am. Chem. Soc. 122, 9344-9345.

PT

¨ [46] Lachmann, P., Huang, Y., Reimann, J., Flock, U., Adelroth, P. (2010) Substrate control of internal electron transfer in bacterial nitric oxide reductase, J. Biol. Chem.

AC CE

285, 25531-25537.

[47] Hendriks, J.H., Jasitis, A., Saraste, M., Verkhovski, M.I. (2002) Proton and electron pathways in the bacterial nitric oxide reductase, Biochemistry 41, 2331-2340. [48] Daskalakis V., Ohta, T., Kitagawa, T., Varotsis C. (2015) Structure and properties of the catalytic site of nitric oxide reductase at ambient temperature, Biochim. Biophys. Acta 1847, 1240-1244. [49] Blomberg, L.M., Blomberg, M.R.A. and Siegbahn, P.E.M. (2006) A Theoretical Study on Nitric Oxide Reductase Activity in a ba3 -type Heme-Copper Oxidase, Biochim. Biophys. Acta 1757, 31-46.

31

ACCEPTED MANUSCRIPT

[50] Pinakoulaki, E., Ohta, T., Soulimane, T., Kitagawa, T., Varotsis C. (2005) Detection of His-Heme Fe2+ -NO Species in the Reduction of NO to N2 O by ba3 -Oxidase from Thermus thermophilus J. Am. Chem. Soc. 127, 15161-15167. ¨ [51] T. Hayashi, I.-J. Lin, Y. Chen, James A. Fee, and P. Moe`I´Lnne-Locco (2007) Fourier Transform Infrared Characterization of a CuB -Nitrosyl Complex in Cytochrome ba3

PT

from Thermus thermophilus: Relevance to NO Reductase Activity in Heme-Copper Terminal Oxidases, J. Amer. Chem. Soc. 129, 14952-14958.

RI

[52] Riistama, S., Puustinen, A., Garcla-Horsman, A., Iwata, S., Michel, H., Wikstr¨om, M. (1996) Channelling of dioxygen into the respiratory enzyme, Biochim. Biophys. Acta

SC

1275, 1-4.

NU

[53] Riistama, S., Puustinen, A., Verkhovsky, M.I., Morgan, J.E., Wikstr¨om, M. (2000) Binding of O2 and Its Reduction Are Both Retarded by Replacement of Valine 279 by Isoleucine in Cytochrome c Oxidase from Paracoccus denitrificans, Biochemistry 39,

MA

6365-6372.

[54] Funatogawa, C., Li, Y., Chen, Y., McDonald, W., Szundi, I., Fee, J.A., Stout, C.D.,

ED

Einarsdottir, O. (2017) Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase, Biochemistry 56,

PT

107-119.

[55] Hemp, J., Robinson, D.E., Ganesan, K.B., Martinez, T.J., Kelleher, N.L., Gennis,

AC CE

R.B. (2006) Evolutionary Migration of a Post-Translationally Modified Active-Site Residue in the Proton-Pumping Heme-Copper Oxygen Reductases, Biochemistry 45, 15405-15410.

¨ [56] Karpefors, M., Adelroth, P., Namslauer, A., Zhen, Y., Brzezinski, P. (2000) Formation of the “Peroxy” Intermediate in Cytochrome c Oxidase is Associated with Internal Proton/Hydrogen Transfer, Biochemistry 39, 14664-69. ¨ [57] Poiana, F., von Ballmoos, C., Gonska, N., Blomberg, M.R.A., Adelroth, P., Brzezinski, P. (2017) Splitting of the O-O Bond at the Heme-copper Catalytic Site of Respiratory Oxidases, Science Advances 3, e1700279. [58] Brunori, M., Giuffr`e, A., Forte, E., Mastronicola, D., Barone, M.C., Sarti, P. (2004) Biochim. Biophys. Acta 1655, 365-371. 32

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[59] Pinakoulaki, E., Varotsis C. (2008) Nitric oxide activation and reduction by hemecopper oxidoreductases and nitric oxide reductase, J. Inorg. Biochem. 102, 1277-1287. [60] Ohta, T., Soulimane, T. Kitagawa, T., Varotsis C. (2015) Nitric oxide activation by caa3 oxidoreductase from Thermus thermophilus, Phys. Chem. Chem. Phys. 17, 10894-

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 Mechanisms for NO reduction in CcOs with a heme a3 active site are studied.  Free energy profiles are constructed combining computational and experimental data.  Comparisons are made to the mechanism for NO reduction in cNOR.  High reduction potentials in CcOs are found to cause low rates of NO disappearance.  A conserved valine may play a role in reducing the NO reduction rate in CcO.