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Influence of intramolecular secondary sphere hydrogen-bonding interactions on cytochrome c oxidase inspired low-spin heme–peroxo–copper complexes

Structural/electronic influence of metalloenzyme-inspired synthetic heme–peroxo–copper models containing intramolecular hydrogen bonding interactions.


Introduction
In protein-mediated reactions, hydrogen bonding is well known to be critical for active site chemistry, including directing substrate binding and facilitating key catalytic steps. [1][2][3][4][5] This is especially important for metalloenzymes involved in dioxygen processing (such as in O 2 -carriers, oxidases, or oxygenases). For instance, protonation and/or hydrogen bonding is critical in directing O-O bond scission in heme-containing enzymes such as cytochrome P450s, catalases, and peroxidases, by lowering the barrier to O-O reductive cleavage and/or selectively enhancing heterolytic (over homolytic) cleavage. 4,6,7 Catalytic reduction of O 2 to water by heme-copper oxidases (HCOs) involves 8H + and 4e À , wherein 4H + are translocated across the inner mitochondrial membrane, driving ATP synthesis. 5,[8][9][10][11][12] The pertinent mammalian terminal respiratory enzyme is cytochrome c oxidase (CcO), which facilitates O 2 reduction following dioxygen binding to the heterobinuclear active site comprised of a reduced heme (Fe II ) and copper (Cu I ) moiety, initially forming the ferric superoxide species (A, Scheme 1). 5 In the established catalytic mechanism of CcO, the four electrons derive from iron (2e À ; Fe II / Fe IV ), copper (1e À ; Cu I / Cu II ), and a tyrosine residue cross-linked to one His ligand bound to Cu (1e À /1H + ; TyrOH / TyrOc). 5,8,9,11 A unique covalent cross-link between the tyrosine and one of the Cubound histidine residues is believed to modulate both the redox potential and the pK a of the TyrO-H. [13][14][15] It is also postulated that an H-bonding network of water molecules exists in the active site, linking the TyrOH residue to a putative metal-(hydro)peroxide species (I P ), facilitating the necessary proton coupled electron transfer (PCET) from the tyrosine to achieve homolytic O-O cleavage resulting in the formation of the highvalent intermediate P M (Scheme 1). Following a subsequent series of proton (PT) and electron (ET) transfer reactions, also accompanied by membrane proton translocation steps, the initial state of reduced heme and copper (R, Scheme 1) reforms providing for the overall release of two mole-equivalents of water from one mole-equivalent O 2 . [16][17][18][19][20][21][22][23][24] Interestingly, Hbonding interactions involving the active site Tyr have also been shown to be important for controlling proton gating and delivery from the K-channel to the active site for complete protonation-reduction of O 2 to H 2 O. 16 Synthetic heme-peroxo-copper model systems can be strategically designed to probe fundamental chemical questions, including the factors that inuence reductive O-O cleavage, such as H-bonding and/or proton relay processes. These are oen accompanied by electron transfer reactions that result in, and can be applied to, practical catalytic systems; synthetic systems may enhance the understanding of these PT and ET steps to better grasp aspects that may be detrimental to efficiency or longevity. Results from biochemical and computational studies on HCOs, as well as various synthetic hemeperoxo-copper systems, have provided signicant insights into Nature's rationale for taking advantage of hydrogen bonding to easily impart signicant electronic or geometric alterations to tune the enzymatic active site to facilitate O 2 reduction chemistry (heterolytic vs. homolytic cleavage) at metal sites, a biochemical process critical to all aerobic organisms. 17,18,[25][26][27] However, a deeper understanding of how H-bonding impacts O-O cleavage in HCOs is needed, as parts of this mechanism have remained elusive due to the fast kinetics of the homolytic cleavage step (from A to P M ) in the enzyme, which for example has prevented thorough characterization of a widely proposed 2e À -reduced peroxo level intermediate 20,28,29 (I P in Scheme 1, which has recently been supported by experimental and computational studies by Brzezinski, Blomberg, and coworkers) 30 (3) further the development of a more biomimetic, CcO-inspired, synthetic model in order to do corresponding reactivity studies. The results presented herein provide insights into how the incorporation of intramolecular H-bonding moieties affect the electronic structure and core vibrations of the observed low-spin heme-peroxocopper complexes. In most cases, density functional theory calculations of the energy-minimized structures accompany the spectroscopic observations and lend support for the conclusions drawn.

Development of synthetic hydrogen bonding complexes
Mechanistic insights into O 2 activation and reduction from promising reactivity studies of heme-peroxo-copper synthetic systems utilizing the copper ligand TMPA (and also a porphyrinate with a tethered TMPA moiety; TMPA ¼ tris(2pyridylmethyl)amine) have been reported. 32 )-Cu II (TMPA)] + , (DCHIm ¼ 1,5-dicyclohexylimidazole) in 2-methyltetrahydrofuran (MeTHF) solvent under cryogenic conditions (À90 C, Fig. 1). Most heme-containing metalloenzymes that perform dioxygen chemistry (e.g., catalases, peroxidases, P450's, oxidases, and globins) require a proximal axial base; hence, to generate low-spin species and understand the H-bonding interactions in these heme-based metalloenzyme mimics, the strong electron donating axial base, DCHIm, was employed, mirroring the histidine proximal ligand in CcO. Novel low-spin heme-peroxo-copper synthetic complexes using eight TMPA-derivatized copper ligands ( Fig. 1) have been prepared in order to develop a series of compounds that have the potential or do in fact demonstrate signicant H-bonding capabilities; H-bonding has previously been shown in copper(II) chloride complexes bearing some of these ligands. 44 A systematic study presented here enables us to examine the effects of secondary coordination sphere substitutions on the structural and electronic properties such as (1)  As such, TMPA-based copper ligands, with various substituents in the 6-pyridyl position, were thoughtfully synthesized and utilized: CH3 TMPA, (CH3)2 TMPA, OCH3 TMPA, PV TMPA, A2 TMPA, NH2 TMPA, (NH2)2 TMPA, and NH2,CH3 TMPA ( Fig. 1; see ESI † for synthetic and characterization details). In particular, comparison of heme-peroxo-copper complexes bearing the ligands NH2 TMPA, CH3 TMPA, and OCH3 TMPA can help differentiate steric vs. H-bonding inuences; we can rule out electrondonating effects on the basis that the Cu II/I reduction potential of complexes bearing these ligands are very similar (E 1/2 ¼ À0.34 or À0.35 V) (Fig. S3 & Table S2 †), indicating the three ligands donate electron density to the copper ion in a nearly identical manner. § Another important property is revealed by the present study and data obtained; the metal-oxygen stretching frequencies (especially Cu-O) are typically not observed via resonance Raman spectroscopy (rR) in previously studied synthetic heme-peroxo-copper complexes. 36,39,[45][46][47][48] However, as demonstrated herein (vide infra), we have found that systems with the TMPA infrastructure yield strong resonance enhancement of both the Fe-O and Cu-O stretches, offering unique and valuable insights into the bonding in the heme-peroxo-copper core.

Azido-Cu(II) complexes to assess ligand H-bonding capabilities
To assess the H-bonding capabilities of these ligands, the azido complexes, [( X TMPA)Cu II (N 3 À )] + , many of which were previously reported and characterized by X-ray crystallography by Masuda and coworkers (Fig. 2), 49,50 were prepared and, for the rst time, analyzed by IR spectroscopy to observe steric, electronic, or Hbonding inuences on the vibrational stretching frequency characteristic of the N 3 À moiety ( Fig. 3 and S2 and Table S1 †).
Substitution of an amino moiety on the TMPA framework, [( NH2 TMPA)Cu II (N 3 À )] + , vs. the parent TMPA azido complex showed that the former possesses a higher n(N-N) stretching frequency by 10 cm À1 ( Fig. 3A    As evidenced by comparing the n(N-N) values for the cupric azido complexes bearing the ligands NH2 TMPA and TMPA (Fig. 3A) vs. NH2,CH3 TMPA and CH3 TMPA (Fig. 3B), the NH2 TMPA and NH2,CH3 TMPA ligands have the same H-bonding capabilities (each shows a 10 cm À1 blue-shi when compared to their respective "base" ligand). The azido derivative with CH3 TMPA has a n(N-N) value slightly higher than with TMPA, implicating a negligible steric inuence ( Fig. S2A & Table S1 †). The PV TMPA cupric azido complex has a n(N-N) stretching frequency similar to [( (NH2)2 TMPA)Cu II (N 3 À )] + (within 1 cm À1 , Fig. S2B & Table S1 †), suggesting that one pivalamido group can exert an H-bonding strength roughly equivalent to two -NH 2 groups. Masuda and coworkers also previously showed that some of the same H-bonding copper ligands utilized herein ( PV TMPA, NH2 TMPA, and (NH2)2 TMPA) provide for enhanced stability of dicopper(II) trans-peroxide complexes complexes (Fig. 4) due to H-bonding to the peroxo core. 49,50 Further, PV TMPA has also been shown to stabilize a cupric superoxide complex (vide infra). 51 Thus, we hypothesized that amino functionalities may stabilize a heterobinuclear peroxide complex as well, and thereby generate good systems to carry out future reactivity studies; one would expect the barrier to reduce the O-O bond would be less if H-bonding caused bond elongation. Therefore, these interactions may facilitate O 2 -reduction chemistry by these systems. Ligands bearing methyl ( CH3 TMPA & (CH3)2 TMPA) or methoxy substituents (newly synthesized OCH3 TMPA) ( Fig. 1) were included in the series to serve as appropriate controls, wherein the methyl groups or methoxy group may inuence the Cu-geometry in a similar manner as in the other derivatives, however without any hydrogen bonding interaction. The mixed derivative, NH2,CH3 TMPA, was newly synthesized (Fig. S1 †) § to probe whether both amino moieties in LS-(NH2)2 TMPA interact with the O-O peroxo core (and to assess if the second amino group doubles the magnitude of the H-bonding effects or interacts to a lesser degree).
Results collected in this work, further described below, (1) demonstrate the balance between sterics and H-bonding in developing appropriate synthetic systems to model behaviors featured in metalloenzymes, (2) establish the importance of corresponding controls in verifying novel synthetic models, and (3) show evidence supporting the H-bonding capabilities of the CcO-inspired low-spin heme-peroxo-copper complexes bearing the ligands NH2 TMPA, (NH2)2 TMPA, and NH2,CH3 TMPA.

Synthesis and UV-vis spectroscopic characterization
The spectroscopic properties of the title compounds are evaluated in the context of established (heme-)copper chemistry. 26 )-Cu II ( X TMPA)]BArF (LS-X TMPA, Fig. 1 and 5) which are the focus of this work as most heme metalloenzymes can only function with (and depend on) a proximal axial base. 5,6,28 The binding mode of the peroxide ligand in the parent HS-TMPA complex has been previously well established to  possess a m-h 2 :h 1 (side-on to iron, end-on to copper) bridge ( Fig. 5), based on a great deal of spectroscopic data, DFT calculations, and precedent from an early X-ray structure of an analog compound characterized by Naruta and coworkers. 33,40,57,58 The high-spin state of the parent heme-peroxo-copper complex HS-TMPA (overall S ¼ 2) was also previously conrmed by the rR spin state marker band, n 2 , 31 by 2 H-NMR spectroscopy (observation of a paramagnetic peak using pyrrole-deuterated F 8 porphyrin) 59 and via EPR spectroscopy (the observed X-band silent spectrum was ascribed to antiferromagnetic coupling between the iron and copper ions). 59 The peroxo binding mode in the low-spin heme-peroxo-copper complex LS-TMPA (Fig. 5) was previously determined to be endon to both copper and iron (trans-m-h 1 :h 1 ) based on the combination of spectroscopic data, DFT calculations, and analogy to previously characterized low-spin species. 36 Based on DFT energy-minimized structures for HS-vs. LS-TMPA, 33,37,59,60 the end-on formulation in the low-spin species also results in an elongated metal-metal distance (HS-TMPA: r(Fe/Cu) ¼ 3.982Å )-Cu II ( X TMPA)] + were newly generated at À90 C in MeTHF (structures depicted in Fig. S4 †), and subsequent UV-vis monitoring of their formation indicated they all had comparable spectra with a Soret $416 nm and a split Q-band l max $ 560 nm and a diagnostic $650 nm absorption feature (a porphyrin to iron transition oen called CT2 in heme literature) 61 regardless of the TMPA substituent (Table 1, Fig. S5 and S6 † in blue). Due to the comparable UV-vis spectra of these HS-X TMPA complexes, Fig. S4-S6 †), the paramagnetic 2 H-NMR shis observed (Fig. S7 † in blue), and their silent EPR spectra, these new species were conrmed to be high-spin. It is of note that the similar UV-vis spectra, paramagnetic 2 H-NMR signal, and silent EPR spectra (X-band) are consistent with many other high-spin heme-peroxo-copper complexes previously reported (not just with HS-TMPA). 27,36,53,59,62 The UV-vis spectra show a red shi in both the Soret ($7-12 nm) and Q-band ($27 nm) aer addition of one equivalent of DCHIm (Fig. 5) to the HS-X TMPA complexes ( Fig. S5 and S6 † in green), consistent with previously reported shis of synthetic heme-peroxo-copper complexes going from HS to LS-X TMPA species. 26,36,62 Thus, with eight new HS (Fig. S4 †) and then eight corresponding LS complexes, a total of sixteen new heme-peroxo-copper species are described here. However, this work focuses particularly on the imidazole-ligated low-spin complexes, which possess a heme structure comparable to the CcO binuclear active site (Fig. 1). The low-spin state (S ¼ 0) of these novel LS-X TMPA complexes was conrmed by the rR spin state marker band, n 2 (Table 1) thus the observed signal is from the pyrrole deuterons, Fig. S7 † in green), and from silent EPR spectra being observed for all the complexes, indicating antiferromagnetic coupling between the low-spin iron(III) and copper(II) sites, mediated by the peroxo bridging ligand. These ndings are consistent with the previous spectral characterization of the parent heme-peroxo-copper complex LS-TMPA. 36,40,59 The UV-vis spectral changes from the high-spin to low-spin species in each case are analogous to those reported for similar model complexes (vide infra), which have been shown to be concomitant with a change in binding mode from m-h 2 :h 1 to trans-m-h 1 :h 1 . 26,27,33,36,37,59,60 Therefore, we propose at this time that the core peroxo binding mode in the novel LS-X TMPA complexes is also end-on coordinated to both metal ions, as depicted in Fig. 1 and 5.
All of the LS-X TMPA complexes have similar UV-vis spectral features with a Soret absorption around 425 nm and a Q-band l max around 533 nm regardless of the Cu ligand identity  azido and dicopper(II) trans-peroxo complexes bearing the ligands NH2 TMPA and (NH2)2 TMPA). 49,50 This is attributed to the H-bond interaction lowering the peroxo p* donor orbital energies more than the Fe acceptor orbitals (see Fig. 6, S6 † & Table  1). Importantly, this diagnostic marker, the blue shi of the low energy absorption l max , increases systematically with the number of -NH 2 substituents (Fig. 6, S6 † & Table 1), providing compelling evidence that all -NH 2 groups employed interact with the peroxo core. In line with these trends, LS-NH2,CH3 TMPA shows low energy features similar to LS-NH2 TMPA, while those of LS-(NH2)2 TMPA are shied to even higher energy, suggesting that both amino groups of LS-(NH2)2 TMPA are H-bonding to the O-O peroxo core and that it is not due to the additional sterics from incorporating a second functionality on the TMPA framework (Fig. 6, S6 † & Table 1). These conclusions about H-bonding to the peroxo core by the ligand-amino pendants are supported by resonance Raman spectroscopic interrogation (vide infra).
Masuda and co-workers 49 previously demonstrated increased H-bonding capabilities from PV TMPA (with pivalamido potential H-bonding moiety (Fig. 1)) relative to NH2 TMPA by comparing their respective dicopper(II) trans-peroxide complexes; this is expected since -pivalamido groups are more acidic than -amino substituents. Thus, to optimize the amount of H-bonding interactions between the intramolecular functionality and the O-O peroxo core, the low-spin heme-peroxocopper complex with PV TMPA (i.e., [(DCHIm)(F 8 )Fe III -(O 2 2À )-Cu II ( PV TMPA)]BArF, LS-PV TMPA) was synthesized (Fig. 1). The UV-vis spectrum exhibits similar Soret (429 nm) and Q-band l max (533 nm) absorptions as all the other LS-X TMPA derived complexes (Table 1), however, the low-energy region exhibits uniquely one broad intense peak around 840 nm (Fig. S5G, S6G † & Table 1). While this effect could indicate H-bonding to the peroxo core, it is notable that this low-energy band for LS-PV TMPA is markedly more intense than observed for the other LS-X TMPA complexes (3 $ 6000 mM À1 cm À1 vs. 2000-4000 mM À1 cm À1 ). This deviation may indicate a structural change (or distortion of the dihedral Fe-O-O-Cu angle), that is likely imparted by the presence of the very bulky tert-butyl group. To probe this possibility, a new ligand with a less bulky carbonyl substituent (a methyl instead of a tert-butyl group) was synthesized ( A2 TMPA, with a 6-pyridyl -acetylamido moiety) and the corresponding DCHIm-bound low-spin heme-peroxocopper complex was prepared (Fig. 1). The UV-vis spectrum of LS-A2 TMPA) was similar to LS-PV TMPA, exhibiting one broad, intense low-energy feature around 823 nm (Fig. S5F, S5G, S6F and S6G, † Table 1). This indicates that the amido functionality impacts the peroxo core in a manner that is distinct from amino or methyl groups alone, potentially arising from variation in the geometric and electronic structure around the Cu (vide infra). This nding demonstrates the careful balance between sterics and electronics (i.e., H-bonding) in our synthetically derived heme-peroxo-Cu complexes (highlighting the intricacies in developing synthetic models), which nature has also mastered a See the ESI for the UV-vis spectra ( Fig. S5 and S6) and for the ( 16 O 2 -18 O 2 ) data and raw rR spectra (Fig. S8). in its design of metalloenzymes in order to dictate biologically required catalytic transformations. These diagnostic low-energy CT bands (Fig. 6 & S6 † and Table 1) provide valuable evidence for how variations in Cu ligand substitution inuence the peroxo core and offer indirect evidence for intramolecular H-bonding interactions for the three novel low-spin heme-peroxo-copper complexes containing amino groups (LS-NH2 TMPA, LS-(NH2)2 TMPA, and LS-NH2,CH3 TMPA, Fig. 1), and potentially two amido-containing complexes (LS-PV TMPA and LS-A2 TMPA, Fig. 1). As other research labs, such as those of Masuda, Nocera, and Dey, 50,[64][65][66][67] have demonstrated inter-and intramolecular H-bonding in dicopper-peroxide and heme (hydro)peroxide complexes through the use of resonance Raman spectroscopy, we have therefore also evaluated our new low-spin heme-peroxo-copper complexes (Fig. 1) using rR spectroscopy and corresponding computational analysis (vide infra).

Impact of H-bonding and sterics on M-O and O-O bonds in the peroxo core as determined from rR spectroscopy
Insight into the effect of these H-bonding interactions is provided by rR spectroscopy, which can serve as a valuable probe of O-O and M-O bond strengths, and distinguish between H-bonding to the proximal or distal O-atom (with respect to Fe). 26,27,68,69 Importantly, we nd that signicant differences in the rR data for these low-spin complexes are observed (vide infra) as a consequence of the derivatization of the TMPA ligands as outlined in Fig. 1.
Among both high-spin and low-spin synthetic model complexes compiled from 15 reports in the literature, a Cu-O stretch has been observed in only three LS species out of 24 HS and LS complexes. 36,39,45,46,48,53,55,56,59,[70][71][72][73][74][75] Further, of those three, only one employed a copper ligand that was not TMPA (the other two being the HS and LS parent TMPA complexes). 36,59,72 Thus, this report more than doubles the description and characterization of known synthetic low-spin heme-peroxo-copper complexes in the literature, with 8 newly prepared species. The remarkably strong resonance enhancement of both the Fe-O and Cu-O stretches with the TMPA ligand may be due to vibrational coupling and/or the presence of an LMCT band masked by the Soret absorption used for excitation. Thus, the rR data on these complexes with derivatized TMPA ligands afford critical insights into the nature of the O-O peroxo core.
It is highly relevant to note that in order to mimic the unique tyrosine-histidine crosslink coordinated to the copper ion in CcO, the Karlin and Naruta labs, separately, have reported on synthetic heme-peroxo-copper complexes with a phenolimidazole crosslink moiety (Fig. 7). 70,74 Interestingly, no Hbonding interactions were observed between the phenol and peroxo core, based on rR spectroscopic data (i.e., no effect on the O-O vibrational frequency was observed). This would likely also be the case in the CcO active site, where the nearby tyrosine residue is too far away ($6Å) to directly H-bond to the O-O peroxo core of a putative I P intermediate (Scheme 1). However, an H-bonded network of interstitial water molecules has been suggested, in many computational studies, 28,76,77 to provide the means for proton transfer (from tyrosine) and to facilitate O 2 reductive cleavage. Addressing the growing interest in understanding possible interactions with such active site water molecules, this study presents the rst demonstration of intramolecular hydrogen bonding in a synthetic heme-peroxocopper model system incorporating H-bonding groups.
As mentioned, signicant differences were observed in the rR data ( Fig. 8 and S8 † and Table 1){ for these low-spin complexes with derivatized TMPA ligands (i.e., LS-XTMPA,    Table 1). In contrast, substitution of an H-bonding amino group into the complex (i.e., LS-TMPA vs. LS-NH2 TMPA, LS-NH2,CH3 TMPA) appears to minimally impact the Fe-O and Cu-O bond strengths, while greatly weakening the O-O bond. This is a particularly important nding, as a similar decrease in the n(O-O) vibrational frequency was observed for an H-bonded adduct between a heme-peroxo-Cu model complex and an exogenous phenol (a shi from 870 to 827 cm À1 upon addition of p-nitrophenol, Scheme 2). 26 The differences observed between LS-CH3 TMPA and LS-NH2 TMPA are attributable to H-bonding in the latter, since there is no electronic difference between the two ligands (as determined from Cu II/I reduction potentials, as mentioned above §). Interestingly, incorporating a second H-bonding group (as in LS-(NH2)2 TMPA) acts to weaken all three core modes ( Fig. 8 and S8D, † & Table 1 Table 1). The less sterically hindered amidosubstituted complex LS-A2 TMPA nevertheless yielded similar rR data to LS-PV TMPA (Fig. S8F † and Table 1), suggesting that these two complexes impact the peroxo core in a comparable manner. However, the rR data obtained for LS-OCH3 TMPA (steric control bulkier than CH3 TMPA) are remarkably similar to LS-PV TMPA and LS-A2 TMPA, including isotope-sensitive modes as well as heme bands (Fig. S8H † and Table 1), suggesting that the effects of the PV TMPA ligand on the peroxo core involve little or no H-bonding, but are predominantly steric in nature.
Further, valuable information is provided by resonance Raman data proled over the low energy (700-900 nm) region (with excitation at various wavelengths, Fig. S9 †). In particular, the complexes LS-(NH2)2 TMPA and LS-NH2,CH3 TMPA show preferential enhancement of the n(Fe-O) and n(O-O) modes at lower energy excitation, with enhancement of the n(Cu-O) and n(Fe-O) (and some n(O-O)) observed at higher energy excitation. This indicates the presence of two distinguishable LMCT absorption bands (illustrated for LS-(NH2)2 TMPA in Fig. S10 †), wherein the lower energy transition involves excitation from a peroxo orbital that is donating primarily into Fe, while the higher energy transition involves a peroxo orbital donating into both metals. While the remaining complexes studied herein did not exhibit rR proles indicating two distinct bands, it is worth noting that the complexes LS-PV TMPA and LS-A2 TMPA yielded exceptionally strong n(Fe-O) and n(O-O) enhancement, but very weak Cu-O enhancement, suggesting primarily O 2 2À to Fe CT character.
It is valuable to note that our analysis leading to the conclusion that the pivalamido group in LS-PV TMPA does not or only weakly H-bonds to the peroxo bridging ligand (and see the DFT calculations, below) stands in strong contrast to what we know for the cupric superoxo complex (without any heme present) with the PV TMPA ligand, Fig. 9.

Electronic insights from DFT calculations: orbital considerations for H-bonding effects
In order to better understand how hydrogen bonding impacts the heme-peroxo-copper core, DFT calculations were performed to correlate to the spectroscopic data. Geometry optimization for LS-TMPAk and LS-NH2 TMPA reveals distinct changes in the O-O, Fe-O, and Cu-O bond lengths that provide good agreement with the trends observed in the rR data (Table  1). Analytical frequency calculations support the observed trends, although vibrational modes are oen highly mixed in the calculations. Therefore, predicted changes in vibrational frequency were also computed based on calculated structural changes using Badger's Rule (Table S3 †), providing a direct correlation between bond length and stretching frequency for two-atom-centered vibrational modes.
Scheme 2 Previously reported hydrogen-bonding adduct that formed upon addition of 10 equivalents of 4-nitrophenol to a low-spin heme-peroxo-copper complex. Resonance Raman results reported were consistent with the rR in this work (Table 1), where a decrease is observed in the n(O-O) when H-bonding groups interact with the peroxo core. To evaluate how the -NH 2 group in LS-NH2 TMPA interacts with the peroxo core, four principle orientations were considered, wherein the H-bond is directed toward either O Cu (Fig. 10A and 10B) or O Fe (Fig. S10A Table 1), indicating that the bonds weaken and elongate. This behavior can be replicated computationally by constraining and systematically elongating the Fe/Cu distance in LS-TMPA, which results in weakening of all three core modes, therefore suggesting that the steric consequence of the methyl group in CH3 TMPA is to push the Fe and Cu moieties apart.
However, incorporating substituents with greater steric contributions, as in the amido-based groups of PV TMPA and O-O bond lengths compared to LS-PV TMPA, in excellent agreement with the trend in vibrational frequencies from the rR data), suggesting that the H-bond in these amido complexes has a minor impact on the core bond strengths. Offering insight into the intense low-energy band observed by UV-Vis for LS-PV TMPA and LS-A2 TMPA (vide supra), the calculations also show that H-bonding to the O 2 p* s orbital polarizes the O 2 p* v orbital toward the O Fe atom, which would result in a more intense LMCT band. Taken together, these results suggest that the effect of H-bonding to the peroxo core in the amidoappended complexes differs from the amino-appended complexes by a combination of structural and electronic aspects.

Conclusions
Hydrogen bonding is prevalent in biology, especially concerning dioxygen chemistry in metalloenzymes (i.e., globins, peroxidases, catalases, cytochrome P450's, oxidases). 6 The effects of intramolecular H-bonding will allow for interesting mechanistic studies for reductive O 2 cleavage; this could be invaluable in the design of more efficient fuel cells in which O 2 reduction is the key reaction occurring at the cathode. This study has fully characterized eight novel synthetic low-spin heme-peroxo-copper models inspired by cytochrome c oxidase active site chemistry; overall 16 complexes have been newly synthesized, which more than doubles reported analogous complexes in the literature.
The present work demonstrates the exibility to design and synthesize various novel TMPA-based ligands and observe a range of effects (elucidated here), reinforcing the importance of a careful balance between sterics and hydrogen bonding being critical in enzyme active site reactions, those requiring specic H-bonding or protonation situations. This is especially shown using the ligands containing appended amido moieties, which have been shown to be strong H-bonding groups in previous studies (with copper-only complexes) but herein perturb the heme-peroxo-copper complex geometry, hindering signicant H-bonding to the peroxo core. Note that the Cu geometry is now skewed toward square pyramidal (s 5 ¼ 0.22 and 0.16 for (A) and (B), respectively), unlike the optimized structures for LS-NH2 TMPA (Fig. S11 †). Further, note the distorted dihedral angle of 100 and 107 (for (A) and (B), respectively) vs. 141 for LS-TMPA (Fig. S13A †).
The effects of intramolecular H-bonding on the heme-peroxo-Cu core were examined by UV-vis and rR spectroscopies and DFT calculations. It is shown that the H-bonding moieties (-NH 2 , -(NH 2 ) 2 , and -NH 2 ,CH 3 ) reproduce experimental data for intermolecular H-bonding effects reported for a related heme-peroxo-copper systems, and preferentially interact with the O Cu atom of the peroxo; these ndings offer a critical correlation to studies showing that an H-bonding interaction can lower the O-O cleavage barrier. 26,27 Importantly, while the nature of the H-bonding interaction could not be well understood in past studies due to complications including poorly dened Cu ligation and entropic costs, the ligand system employed herein provides a well-dened platform that is reliably modelled by computational means. This has delivered critical insight into an orientational anisotropy in H-bond activating the O-O bond for cleavage. Furthermore, while the metal-oxygen bond vibrations (especially Cu-O) are not observed for most synthetic heme-peroxo-copper species, these low-spin complexes for the rst time allowed direct insight into the Cu-O bond; this is a paramount step towards future development of a more biomimetic, efficient model of CcO (with full access to modications of new ligand systems and a deeper understanding of what is occurring during O 2 reduction studies). Overall, systematic control over H-bonding ability of the X TMPA ligands provides a foundation to draw conclusions about the effects of H-bonding on the thermodynamic stability, electronic structure, and reactivity of corresponding hemeperoxo-copper complexes.

Conflicts of interest
There are no conicts to declare.