Proton-coupled electron transfer reactivities of electronically divergent heme superoxide intermediates: a kinetic, thermodynamic, and theoretical study

Heme superoxides are one of the most versatile metallo-intermediates in biology, and they mediate a vast variety of oxidation and oxygenation reactions involving O2(g). Overall proton-coupled electron transfer (PCET) processes they facilitate may proceed via several different mechanistic pathways, attributes of which are not yet fully understood. Herein we present a detailed investigation into concerted PCET events of a series of geometrically similar, but electronically disparate synthetic heme superoxide mimics, where unprecedented, PCET feasibility-determining electronic effects of the heme center have been identified. These electronic factors firmly modulate both thermodynamic and kinetic parameters that are central to PCET, as supported by our experimental and theoretical observations. Consistently, the most electron-deficient superoxide adduct shows the strongest driving force for PCET, whereas the most electron-rich system remains unreactive. The pivotal role of these findings in understanding significant heme systems in biology, as well as in alternative energy applications is also discussed.


Introduction
Activation of dioxygen by heme proteins plays a pivotal role in metalloenzyme-mediated oxidation, oxygenation, and dioxygen reduction reactions in biology. 1 The central paradigm of this dioxygen binding and activation process by heme centers embodies a distinctive panel of intermediates, where the stepwise reduction of O 2 leading up to O-O bond cleavage occurs in parallel to oxidation of the heme iron center.The initial hemedioxygen adduct (i.e., heme-superoxo (Fe III -O 2 À c) or heme-oxy (Fe II -O 2 ) species) is common to all dioxygen activating heme enzymes, and exhibits a remarkably divergent reactivity prole mainly depending upon the intricate structural tuning within a given active site.Specically, the identity and properties of the amino acid side chain ligating at the heme proximal site, distal and proximal non-covalent interactions about the heme center, and electronic properties of the heme ligand itself, 1a,2 all of which, in concert choreograph the specic biological role of a heme superoxide intermediate.These include implications in (Chart 1): (i) reversible dioxygen binding in hemoglobin (Hb) and myoglobin (Mb); 3 (ii) indole dioxygenation reactivity in tryptophan and indoleamine 2,3-dioxygenases (TDO/IDO); 4 (iii) indole monooxygenation by MarE; 5 (iv) interaction with physiologically present nitric oxide (cNO (g) ) to generate heme peroxynitrite (Fe III -OONO) species in tryptophan nitrating TxtE 6 and other proteins; 7 (v) reactivity with electrons and/or protons in native mechanisms of heme oxygenase (HO), 1b,8 cytochrome P450 (Cyt P450), 9 aromatase, 10 and nitric oxide synthase (NOS); 11 (vi) abstraction of a hydrogen atom in one of the proposed mechanisms of nitric oxide synthase.1a,12 Particularly, heme superoxide reactivities with exogenous electrons and/or electrons and protons are central to an array of heme enzymes, where the site of reduction and/or protonation is critical for the overall outcome of the biological function.For example, reduction followed by protonation of the distal oxygen atom with respect to the iron center gives rise to the corresponding heme hydroperoxide species (e.g., Cyt.P450), 9d whereas the protonation at the proximal oxygen has been oen shown to liberate protonated superoxide, as in the case of unproductive degradation of oxyhemoglobin to met-hemoglobin. 13etailed physicochemical elucidation of reduction-protonation chemistries of heme superoxide adducts is also of supreme interest with regard to the oxygen reduction reaction (ORR), a cornerstone in numerous critical biological (e.g., cellular respiration and oxidative phosphorylation) and industrial (e.g., synthetic catalysis and batteries) processes. 14In that, whether the heme superoxide abstracts a hydrogen atom (Hc ¼ H + + e À ) in a single mechanistic step (i.e., concerted), or an electron and a proton (or vice versa) in two consecutive steps (i.e., stepwise) is salient in dictating the overall thermodynamics of the reaction landscape.All of these concerted and stepwise mechanistic possibilities fall under the umbrella of proton-coupled electron transfer (PCET) reactions, 15 of which, the precise mechanistic details are solely dependent on a few key thermodynamic parameters (vide infra). 16Recent thorough investigations by Mayer and coworkers have underscored the importance of PCET processes of heme superoxide adducts with relevance to the cathodic reaction in fuel cells, where efficient, cheap dioxygen reducing metallocatalysts could be of momentous importance. 17Specically, the proton affinity of the hemebound superoxide moiety is critical as it dictates the overpotential barrier of the ORR catalyst, thereby determining the rate/efficiency of the catalytic process.17b, 18 Essentially, the pK a of the protonating acid is climacteric for the overall catalytic outcome as it should deliver weakly interacting protons that enhance the susceptibility of heme superoxide toward reduction, while preventing dissociation of protonated superoxide; protonated superoxide radicals are highly unstable, and have long known to decay giving O 2 and H 2 O 2 (2HO 2 19 Such subtleties related to pK a of the proton source are also critical further downstream in the ORR pathway, where protonation of proximal or distal O-atom of the heme hydroperoxide dictates whether 2-electron (O 2(g) + 2H + + 2e À / H 2 O 2 ) or 4-electron (O 2(g) + 4H + + 4e À / 2H 2 O) reduction of oxygen is accomplished, respectively. 20Patently, the latter process is of preference for fuel cell applications, where the complete 4electron reduction of O 2(g) to H 2 O engender an efficient cathodic reaction, preventing the generation of partially reduced reactive oxygen species.Suitably, the detailed body of work by Dey and coworkers on bio-inspired ORR electrocatalysis have offered classic examples of how to adapt inexpensive, environmentally benign metallosystems for the efficient reduction of O 2(g) to H 2 O. 20c, 21 Despite the widespread signicance of heme superoxide mediated PCET pathways, a comprehensive fundamental understanding of the exact mechanistic, structural, and thermochemical parameters governing these processes is still lacking.Synthetic small molecule models can be useful tools in this endeavor, where in-depth thermodynamic and kinetic investigations into systematically varied heme structures are more feasible.Nonetheless, heme superoxide mimics are abundantly known as incompetent oxidants, and their directed reactivities with organic substrates are extremely scarce.Indeed, our recent work marks the rst report where synthetic heme superoxide intermediates were shown to react with exogenously added indole substrates in the efficient modelling of tryptophan dioxygenation chemistry of indoleamine and tryptophan 2,3-dioxygenases. 22imilarly, heme superoxide adducts that efficiently react with added acids (i.e., protons (H + )), reductants (i.e., electrons(e À )), and/or Hc donors are only a handful. 23Intriguingly, Naruta, 24 Dey, 25 and their coworkers have presented unique examples of heme superoxide intermediates that react with intramolecular H + or Hc donors, ultimately giving rise to the corresponding heme hydroperoxo (Fe III -OOH) adduct.To the best of our knowledge, recent work by Karlin and coworkers is the only instance where proton-coupled electron transfer reactivities of heme superoxide intermediates have been shown, where Hc abstraction from an exogenous, weak O-H bond substrate generates the corresponding heme hydroperoxo species in a single kinetic step. 26n the present investigation, we have interrogated the concerted proton-electron (i.e., Hc) abstraction reactivities of three electronically different, geometrically similar heme iron superoxo complexes, [(Por)Fe III (O 2 that, the most electron-decient superoxide adduct reacts the fastest with TEMPO-H, while the most electron-rich superoxo species remains unreactive.This reactivity pattern is further corroborated by the experimentally determined (i.e., by means of the Bordwell relationship; eqn (1) 27 ) O-H bond dissociation free energies (BDFEs) of the heme hydroperoxo products.This study marks the rst report where detailed thermodynamic (elucidation of pK a , E , and BDFE values) and kinetic (rate comparisons, kinetic isotope effects and activation parameters) investigations are described for a series of structurally similar, electronically divergent heme superoxide intermediates, along with strong theoretical justication.These ndings are crucial in the unequivocal comprehension of both biological systems that are indispensable in human therapeutic applications, as well as processes central to alternative energy sources such as fuel cells.The PCET reactivities of these heme superoxide oxidants were then evaluated against variable concentrations of the TEMPO-H substrate (BDFE ¼ 66.5 kcal mol À1 in THF).When 100 equiv. of TEMPO-H was added into a solution of [(F 20 TPP) Fe III (O 2 À c)] in THF at À80 C, patent changes in absorption features (Soret: 413 to 415 nm; Q-band: 532 to 530 and 553 nm) were evidenced (Fig. 1A), indicating the Hc abstraction reactivity of [(F 20 TPP)Fe III (O 2 À c)] with TEMPO-H (Scheme 1).Importantly, the Hc here is transferred in a single mechanistic step (i.e., concerted), rather than a H + and an e À in two separate steps.This is due to only H + or e À transfer from TEMPO-H substrate being extremely thermodynamically uphill compared to Hc transfer.)) bonds were tested against the above series of heme superoxo adducts, where no evidence of any reactivity could be observed.2).The addition of TEMPO-H induced an upeld shi in the pyrrole resonances to d pyrrole ¼ À1.2 ppm, which is indicative of the formation of a low-spin (S ¼ 1/2) ferric heme system (Fig. 2). 31his is in excellent agreement with the d pyrrole 2 H NMR resonances observed for the low-spin [(F 8 TPP-d 8 )Fe III (OOH)] species by Karlin and coworkers (d pyrrole ¼ À0.63 ppm).26a The EPR spectrum of the nal reaction mixture of [(F 20 TPP) Fe III (O 2 À c)] and TEMPO-H predominantly consists of an organic radical signal (g ¼ 2.0; attributed to the TEMPOc radical; yield ¼ 82%), which is overlapped with the S ¼ 1/2 Fe III features of the low-spin heme hydroperoxo product complex (Fig. S3 and S4 †).Notably, all synthetic heme hydroperoxo adducts reported to-date consist of low-spin ferric centers, 23a,c,24a,b,25,26,32 which parallels our aforementioned 2 H NMR and EPR characterizations.Moreover, the putative [(F 20 TPP) Fe III (OOH)] product complex exhibited the isotopic-sensitive resonance Raman frequency for n(Fe-O) at 597 (D 18 O 2 ¼ À30) cm À1 (Fig. 3), which is in-line with other heme hydroperoxo species reported thus far.In further effort to characterize the nal [(Por)Fe III (OOH)] products, we have generated authentic [(Por)Fe III (OOH)] complexes for all three heme systems by reduction, followed by protonation of each superoxo complex (Scheme 2).In that, the one-electron reduction of the [(Por)Fe III (O 2 À c)] complex was achieved with 1 equiv. of cobaltocene, leading to the corresponding side-on ferric peroxo species, [(Por)Fe III (O 2 2-À )] À (as would be expected for a ve-coordinate or solventligated heme superoxide complex 1b ).O 2 ¼ À41) cm À1 (Fig. S8 †).Successive addition of 1 equiv. of [LuH]OTf produced new electronic absorption features at 415 nm (Soret, 3 ¼ 1.5 Â 10 5 M À1 cm À1 ), and 553 nm (3 ¼ 1.0 Â 10 4 M À1 cm À1 ) (Fig. 4A; also see Fig. S7 †   values of the same magnitude (4.8-12.1)36a These large (i.e., >2) KIE values present compelling evidence into the rate limiting nature of the homolytic O-H bond cleavage process (and thus, concerted Hc transfer from the substrate), rather than only proton or electron transfer being the slowest step (where the reaction rate would linearly correlate with either pK a or E of the substrate, respectively, rather than the BDFE).36b Activation parameters for TEMPO-H oxidation by [(F 20 TPP)Fe III (O 2 À c)] and [(TPP)

Results and discussion
Fe III (O 2 À c)] were determined via Eyring analyses of reaction rates from À70 C to À100 C in THF (Fig. 5B and S11 †).The experimental DH ‡ and DS ‡ activation parameter and the activation energies (DG ‡ ) derived therefrom are listed in Table 1.Similar Eyring analyses for PCET reactivities of a limited, yet diverse group of non-heme superoxo complexes have been reported, all of which are in the same order of magnitude as the heme superoxo values reported herein.35a-c,36a Notably, DH ‡ and DS ‡ for most non-heme superoxides are larger than those of heme superoxide adducts; substantially negative DS ‡ values of the latter suggest highly ordered transition states as one of the possible contributing factors to their sluggish PCET capabilities.Interestingly, Shaik and coworkers found higher activation barriers for Cyt P450 superoxide mediated hydrogen atom abstraction when compared with the corresponding compound-I oxidant. 37These activation parameters also divulge the remarkable inuence of the electronic properties of a heme center on its ability to execute successful Hc atom abstraction.
In that, the most electrophilic superoxide adduct, [(F 20 TPP) Fe III (O 2 À c)], exhibits the smallest activation barrier, and thus, reacts the fastest.To the best of our knowledge, this study marks the rst report where activation parameters are presented for any substrate reactivity mediated by synthetic heme superoxide intermediates, hence impeding any detailed comparisons with like systems.Nonetheless, these ndings impart substantial insights into methodologies for further improvement of ORR efficiencies in fuel cells, and mechanistic understanding of human heme proteins with signicant pathological and/or therapeutic value.For example, these activation parameters could shine light on important structural properties of heme ORR catalysts that could be modulated in order to optimally adjust the activation barriers for precise ORR-related reduction and/or protonation events.Comprehending these observed reactivity disparities on thermodynamic grounds calls for experimental examination of O-H bond dissociation free energies (BDFEs) of the [(Por) Fe III (OO-H)] complexes.That is, since the concerted homolytic O-H bond cleavage is the slowest step of the reaction (vide supra), O-H bond strength of the [(Por)Fe III (OO-H)] product complex predominantly dictates the thermodynamic driving force for the overall reaction.These BDFE values can be deduced in a fairly straightforward fashion using the Bordwell equation (eqn (1)), 27 if one could determine the redox potential (E ) and acidity (pK a ) of the metal oxidants that make up the corresponding thermodynamic cycle/square scheme (Scheme 3).16a,29,36a,38 In addition to shedding light on the perceived reactivities, BDFEs also divulge reactivity limitations to be expected based on the thermodynamic portrayal.All of the heme   S2 †) yielded corresponding reduction potentials via Nernst equation (Table S3 †), and were found to be À1.17,À1.18, À1.20 V (vs.Fc +/0 ) for respectively (Table 1).Naruta and co-workers have recently reported theoretical reduction potential values for two tethered axial imidazole coordinated TMP-based heme superoxide systems, which are more negative compared to our values (À1.32 and À1.75 V (vs.Fc +/0 in EtCN)) 24a (Table 1).This trend is consistent with recently reported values for two F 8 TPP based systems by Karlin and coworkers, where the superoxo adduct with tethered axial imidazole coordination displayed a lower reduction potential (À1.33 V) 26b compared to the parent complex (À1.17V) 26a (Table 1).These  electron reduction of [(Por)Fe III (O 2 À c)] adducts by Cr(h 6 -C 6 H 6 ) 2 can be reoxidized stoichiometrically by tris(4-bromophenyl) ammoniumyl hexachloroantimonate ([(4-BrC 6 H 4 ) 3 N]SbCl 6 ; E 1/2 ¼ 0.67 V vs. Fc +/0 in MeCN; 39 Scheme 4) giving the starting superoxide complex, which can then be re-reduced to [(Por) Fe III (O 2 2À )] À by the addition of 5 equiv. of Cr(h 6 -C 6 H 6 ) 2 (Fig. S13 †).

Computational studies
To gain further insight into the intricate electrochemical properties of the iron(III)-superoxo complexes, we did a computational study on both [(F 20 TPP)Fe III (O 2 À c)] and [(TPP) Fe III (O 2 À c)] systems and studied their reactivities with TEMPO-H.The [(TPP)Fe III (O 2 À c)] complex is calculated as an end-on superoxo conguration with an open-shell singlet spin ground state with two unpaired electrons antiferromagnetically coupled in p * xz and p * OO;yz orbitals.The former orbital is a dominant 3d xz atomic orbital on iron in the plane of the FeOO group, while the latter is the antibonding interaction along the O-O bond that also interacts with the 3d yz orbital on iron.The triplet spin state has the same electron conguration but ferromagnetically coupled and its energy with zero-point energy correction is DE + ZPE ¼ 5.6 kcal mol À1 higher in energy.Our calculated ground state; therefore, matches experimental assignments (see above) that the system is EPR silent.Subsequently, we calculated the hydrogen atom abstraction transition states (TS HA ) for the reaction of 1,3 [(TPP)Fe III (O 2 À c)] from TEMPO-H and the optimized geometries are shown in Fig. 7.The reactions are concerted with a single hydrogen atom transfer leading to an iron(III)-hydroperoxo product.The transition states are early with short TEMPO-H distances of 1.075 and 1.057 Å in the singlet and triplet spin states, respectively.At the same time, the accepting O-H distance is long: 1.419 Å in 1 TS HA and 1.473 Å in 3 TS HA .Generally, early transition states correspond with lowenergy hydrogen atom abstraction reactions, while later transition states have much higher energy barriers. 45ydrogen atom abstraction by 1,3 [(TPP)Fe III (O 2 À c)] leads to electron transfer into the p * OO;yz orbital and generates a doublet spin [(TPP)Fe III (OOH)] coupled to a TEMPO radical.This electron transfer is conrmed by the group spin densities that show an increase of spin on the TEMPO group to r Sub ¼ 0.42, while at the same time spin is lost on the dioxygen moiety r OO ¼ 0.69.The hydrogen atom abstraction barrier is DE ‡ + ZPE ¼ 3.2 kcal mol À1 in the singlet spin state and 9.9 kcal mol À1 for the triplet spin state barrier.The low-spin barrier matches the experimentally determined DH ‡ ¼ 2.9 kcal mol À1 excellently.The experimentally determined entropy contribution is relatively large, probably due to a solvent cage surrounding the molecular complex.As shown by previous work of ours, reactions in solution oen have a solvent cage around the active complex that affect entropies and particularly reduces vibrational contributions. 46Because of the fact that the solvent cage was not included in the model, the computational free energy of activation is relatively low and much lower than that observed in experiment as the gas-phase entropy is overestimated.
The imaginary frequency in the transition state is modest: i809 cm À1 in 1 TS HA and i541 cm À1 in 3 TS HA .Typical values for hydrogen atom abstraction imaginary frequencies are of the order of i1200-i1800 cm À1 and implicate a narrow and sharp peak on the potential energy surface. 47The smaller values seen here, also imply that quantum mechanical tunnelling will be less.Indeed, we calculated a KIE ¼ 3.6 using the Eyring model and KIE ¼ 4.0 with the Wigner tunnelling model for the reaction that passes 1 TS HA .By contrast, for hydrogen atom abstraction of aliphatic substrates by P450 compound I or non-heme iron dioxygenases typically values well larger than 12 are calculated. 48Our obtained KIE value is in good agreement with the experimentally determined value of 6.7 (Table 1) and hence the calculations give a similar potential energy landscape and curvature as derived from the experimental work.
Next, we calculated the BDFE of [(TPP)Fe III (OOH)] as the energy difference of its optimized geometry with that of a hydrogen atom and the [(TPP)Fe III (O 2 À c)] complex and nd a value of 61 kcal mol À1 as an energy difference between the singlet spin iron-superoxo and the doublet spin iron(III)hydroperoxo complex and a hydrogen atom (Fig. 8).Our calculated value matches the experimental derivation from redox potential and pK a values well and shows the hydroperoxo O-H bond is relatively weak.Thus, the BDFE of porphyrin/heme ligated iron(III)-hydroxo complexes were calculated for several systems previously.A value of 80.8 kcal mol À1 was obtained for model of horseradish peroxidase that has a porphyrin equatorial ligand and imidazole as axial ligand, while a value of 88.9 kcal mol À1 was found for a P450 model with a thiolate axial ligand. 49As oen the BDFE represents a measurement of the ability of an oxidant to abstract hydrogen atoms efficiently, this means that [(TPP)Fe III (O 2 À c)] will be a weak oxidant and only able to activate substrates with weak C-H or O-H bonds.Indeed, as reported above, our experimental studies show that only reactions with TEMPO-H led to hydrogen atom transfer, while the system is inactive with other aliphatic substrates.These results; therefore, support previous computational studies on the oxidative properties of the iron(III)-hydroperoxo and iron(III)-superoxo intermediates in P450 enzymes that found them to be sluggish oxidants. 37In addition to the BDFE values, we also calculated the one-electron reduction potential (or electron affinity, EA) of the iron(III)-superoxo complex and nd values of 102 and 107 kcal mol À1 for [(TPP)Fe III (O 2 À c)] and [(F 20 TPP)Fe III (O 2 À c)], respectively.Finally, we estimated the gasphase acidity of the iron(III)-hydroperoxo complex (DG acid ) from the experimentally determined ionization energy of a hydrogen atom (IE H ¼ 313.9 kcal mol À1 ) 50 and the difference between EA and BDFE OH .

Conclusions
Despite the ubiquitous nature of proton-coupled electron transfer processes mediated by heme superoxo adducts in both biology and alternative energy applications, understanding of precise reactivity limitations in terms of key physicochemical properties of the heme oxidant is still in its infancy.Variable temperature kinetic (Eyring) studies have allowed the ascertainment of activation parameters (i.e., DH ‡ , DS ‡ , and DG ‡ ) that dictate the aforementioned PCET reactivities of [(Por) Fe III (O 2 À c)] complexes, which are in strong support of their second-order PCET rates.Indeed, computational ndings are in great agreement within the limitations of the employed solvation model.Notably, this study marks the rst report with both experimental and theoretical insights into activation parameters of any substrate reactivity of synthetic heme superoxo systems, which, in this case, could be monumental in the rational design of oxygen reduction catalysts for fuel cell or similar applications.To gain further understanding into the strong inuence of heme center electronics on the competency to abstract an Hc by the ligated superoxide unit, we have determined O-H BDFE's for the entire series of [(Por) Fe III (OOH)] complexes utilizing experimental E and pK a values deduced from redox and acid-base titrations (Table 1), respectively.The O-H BDFE's of 69.1, 67.5, and 66.5 kcal mol À1 found for [(F 20 TPP)Fe III (OOH)], [(TPP)Fe III (OOH)], and [(TMP) Fe III (OOH)], respectively, provide strong thermodynamic evidence in support of the observed limitations in reactivity.That is, [(F 20 TPP)Fe III (O 2 À c)] and [(TPP)Fe III (O 2 À c)] successfully abstracted an Hc from TEMPO-H, while [(TMP)Fe III (O 2 À c)] did not react due to the nullied thermodynamic driving force (i.e., BDFE heme hydroperoxo z BDFE TEMPOH ).Moreover, the trends in experimentally observed O-H BDFE's are unequivocally supported by our computational results.Lastly, this work reveals previously unknown, critical aspects surrounding electronically driven feasibilities of substrate reactivities facilitated by heme superoxo intermediates.This knowledge will broaden the current understanding of long overlooked reactivity properties of mid-valent heme-oxygen intermediates in biology, while offering novel avenues for the design of better ORR catalysts leading to enhanced efficiencies (e.g., ne-tuning of (1) heme systems steered by electronic properties, (2) acidities of proton sources utilized in ORR catalysis, and (3) structure-based thermodynamic (e.g., pK a , E , BDFE) and kinetic (e.g., reaction rates and feasibilities, rate limiting events) properties of heme ORR catalysts to further the overall efficacy etc.).If geometric, electronic, and/or secondary sphere properties of heme superoxide intermediates can be optimized to promote the oxidation of stronger organic substrates is an intriguing unknown, and will be a focus of our future interrogations.

Fig. 1
Fig. 1 Electronic absorption spectral changes observed (in THF at À80 C) during the reaction of a 50 mM solution of (A) [(F 20 TPP)Fe III (O 2 À c)] and (B) [(TPP)Fe III (O 2 À c)] with 100 equiv. of TEMPO-H (red ¼ initial ferric superoxo complex; blue ¼ final ferric product).Insets show the expanded Q-band regions, and arrows indicate the direction of peak transition.

Fig. 3
Fig. 3 Resonance Raman spectra (l ex ¼ 413.1 nm) collected from a 2 mM frozen THF solution of the final heme product from the reaction between TEMPO-H and [(F 20 TPP)Fe III (O 2 À c)] prepared with 16 O 2(g) (black) and 18 O 2(g) (red).
superoxide complexes employed in this study can be quantitatively converted to the corresponding heme hydroperoxo adducts by stepwise reduction-protonation (by cobaltocene and [LuH]OTf, respectively) reactivity as illustrated in Scheme 2. However, to elucidate the relevant E and pK a values experimentally, the equilibrium constants must be determined for each of the reduction and protonation steps.The reduction potential (E ) of the [(Por)Fe III (O 2 À c)]/[(Por) Fe III (O 2 2À )] À redox couple for each system was elucidated by titrating the [(Por)Fe III (O 2 À c)] complexes in THF at À80 C (Fig. 6A and S12 †) with the weak reductant, Cr(h 6 -C 6 H 6 ) 2 (E 1/2 ¼ À1.15 V vs. Fc +/0 in CH 2 Cl 2 ; 39 Scheme 4), to afford equilibrium mixtures of [(Por)Fe III (O 2 À c)] and [(Por)Fe III (O 2 2À )] À .The array of calculated equilibrium constants (Table Scheme 3 Thermodynamic square scheme used to determine the O-H bond dissociation free energies of the [(Por)Fe III (OOH)] complexes in THF, along with the relevant thermodynamic parameters.

Fig. 7
Fig. 7 Optimized geometries of the hydrogen atom abstraction transition states from TEMPO-H by [(TPP)Fe III (O 2 À c)] in the singlet and triplet spin states.Bond lengths are in angstroms, the imaginary frequency in cm À1 and group spin densities (r) in atomic units.The right-hand-side shows the singly occupied molecular orbitals in the singlet and triplet reactants.

[
(TPP)Fe III (O 2 À c)] adducts (Chart 2), respectively, which are in excellent agreement with our theoretical ndings.All heme reactants and products have been fully characterized using electronic absorption, EPR, resonance Raman, and 2 H NMR spectroscopies under cryogenic conditions.Besides, the identities and yields of the resultant [(Por)Fe III (OOH)] complexes have been probed by the comparison of their spectroscopic features with those of the independently prepared (i.e., by reduction-protonation of the [(Por)Fe III (O 2 À c)] counterpart) complexes, and by quantication of hydrogen peroxide liberated upon acidication, respectively.

Table 1
Kinetic and thermodynamic parameters for PCET reactivities of heme and nonheme-superoxo complexes with H-atom donor substrates a See Chart 2 for structures related to this work.b At À80 C. c In THF.d No reactivity was observed.e Fc +/0 ¼ ferrocene/ferrocenium redox couple.f Determined for the corresponding hydroperoxo species.g See reference for experimental conditions.