Superoxo, -Peroxo, and -Oxo Complexes from SPECIAL FEATURE Heme͞o2 and Heme-Cu͞o2 Reactivity: Copper Ligand Influences in Cytochrome C Oxidase Models

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␮ ␮ Superoxo, -peroxo, and -oxo complexes from SPECIAL FEATURE heme͞O2 and heme-Cu͞O2 reactivity: Copper ligand influences in cytochrome c oxidase models

Eunsuk Kim*, Matthew E. Helton*†, Ian M. Wasser*, Kenneth D. Karlin*§, Shen Lu¶, Hong-wei Huang¶, Pierre Moe¨ nne-Loccoz¶, Christopher D. Incarvitoʈ, Arnold L. Rheingoldʈ, Marcus Honecker†, Susan Kaderli†, and Andreas D. Zuberbu¨ hler†

*Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218; ¶Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering at Oregon Health and Science University, Beaverton, OR 97006; ʈDepartment of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716; and †Department of Chemistry, University of Basel, 4056 Basel, Switzerland

Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved February 13, 2003 (received for review November 25, 2002)

II -tet ؍ The O2-reaction chemistry of 1:1 mixtures of (F8)Fe (1; F8 rakis(2,6-diﬂurorophenyl)porphyrinate) and [(LMe2N)CuI]؉ (2; LMe2N (N,N-bis{2-[2-(N؅,N؅-4-dimethylamino)pyridyl]ethyl}methylamine ؍ is described, to model aspects of the chemistry occurring in cytochrome c oxidase. Spectroscopic investigations, along with stopped-ﬂow kinetics, reveal that low-temperature oxygenation of III 1͞2 leads to rapid formation of a heme-superoxo species (F8)Fe - ؊ (O2 ) (3), whether or not 2 is present. Complex 3 subsequently re- ؊ III 2 II Me2N ؉ acts with 2 to form [(F8)Fe –(O2 )–Cu (L )] (4), which thermally III II Me N ؉ converts to [(F8)Fe –(O)–Cu (L 2 )] (5), which has an unusually bent (Fe–O–Cu) bond moiety. Tridentate chelation, compared with CHEMISTRY tetradentate, is shown to dramatically lower the ␯(O–O) values observed in 4 and give rise to the novel structural features in 5.

ioxygen binding to metal ion centers such as copper and iron is of great importance and interest, for fundamental Fig. 1. Structure of the fully reduced (FeIII⅐⅐⅐CuII) bovine cytochrome c oxidase D ⅐⅐⅐ ϭ and practical reasons. In nature, copper and iron proteins structure, Cu Fe 5.1 Å. The diagram was assembled by using PDB ID 1OCR coordinates and the program RASMOL. serve to process O2 in a variety of functions, such as in trans- port of dioxygen in blood, mono- or dioxygenation involving oxygen atom incorporation, and substrate oxidation (dehydro- ␮-␩2:␩2-peroxo-dicopper(II) species, which can be in equilibrium genation or removal of electrons) with concomitant reduction with bis-␮-oxo-dicopper(III) isomers (21, 23, 24). of O2 to hydrogen peroxide or water. Cytochrome c oxidases Thus, with the known tridentate chelation for CuB in cyto- mediate the four-electron four-proton reduction of O2 to water, coupling this exergonic reaction to membrane proton chrome c oxidases, we are intensifying efforts to study such situ- translocation, which drives ATP synthesis (1, 2). Protein x-ray ations in model systems, to see whether O2 intermediates form with FeII͞CuI precursors, and to understand the detailed role of structures reveal that O2 binding and reduction occur at a binuclear active site consisting of a high-spin heme group the copper ligand (tridentate versus tetradentate) in heme- ͞ (with proximal histidine), with a tris-histidine-ligated copper copper O2 chemistry. Here we describe the oxygenation chemis- II Me2N I ϩ ion (CuB) situated on the distal side (see Fig. 1) (3–6). Spec- try of 1:1 mixtures of (F8)Fe (1) and [(L )Cu ] (2). troscopic–mechanistic investigations suggest that after a possi- ble initial O2 interaction with CuB, a myoglobin-like Fe–O2 III Ϫ adduct forms (best described as a Fe –O2 superoxo species), subsequently leading to a FeIVAO (ferryl-oxo) species (7–10), III 2Ϫ II although a prior peroxo-bridged Fe –(O2 )-Cu intermediate has not been ruled out. The tyrosine-crossed-linked CuB site (Fig. 1) is thought to be critical in electron transfer (from CuI and the phenol group) leading to reductive O–O cleavage and proton-translocation chemistry (11–13). Our research program is focused on elucidating the fundamental aspects of O2 interactions with heme and copper centers. A very rich chemistry ensues (Scheme 1), wherein there is an Thus, we have reported several examples where reduced heme– initial rapid formation of a heme-superoxo complex II͞ I ␮ III 2Ϫ Fe Cu complexes react with O2, giving -peroxo Fe –(O2 )– CuII species (14–18). Most of these studies have used tris[2-pyridylmethyl]amine (TMPA) as the Cu ligand. Extensive studies on This paper was submitted directly (Track II) to the PNAS ofﬁce. copper (only) dioxygen systems have shown that even subtle Abbreviations: TMPA, tris[2-pyridylmethyl]amine; F8, tetrakis(2,6-diﬂurorophenyl)porphy- ͞ rinate; LMe2N N,N-bis{2-[2-(NЈ,NЈ-4-dimethylamino)pyridyl]ethyl}methylamine; UV-Vis, changes in ligand structure denticity can dramatically alter the ultraviolet–visible; RR, resonance Raman; THF, tetrahydrofuran; EtCN, propionitrile. I͞ ϭ nature of the (ligand)Cu O2 product [giving Cun–O2 (n 1or ϭ Data deposition: The x-ray data have been deposited in the Cambridge Crystallographic 2) or Cun–(O)2, n 2 or 3] (19–21). Tetradentate TMPA in- Data Centre, www.ccdc,cam.ac.uk (CCDC 206505). II II duces formation of an end-on ligated (Cu –O–O–Cu ) peroxo- §To whom correspondence should be addressed at: Department of Chemistry, Johns I dicopper(II) structure upon O2 reaction with [Cu (TMPA)- Hopkins University, 3400 North Charles Street, Baltimore, MD 21218. E-mail: ϩ (MeCN)] (22), whereas tridentate ligands generate side-on [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0737180100 PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 3623–3628 Downloaded by guest on September 26, 2021 II Me N I ϩ Fig. 2. UV-Vis spectra of the oxygenation reaction of (F8)Fe (1)͞[(L 2 )Cu ] (2) in CH2Cl2͞6% EtCN at 193 K. Shown are an equimolar mixture of 1 and 2 (black); Ϫ III 2 II Me2N ϩ [(F8)Fe –(O2 )–Cu (L )] (4) (red); a product mixture obtained after warming III II Me N ϩ III Scheme 1. 4, which includes [(F8)Fe –O–Cu (L 2 )] (5) and (F8)Fe –OH (green).

Ϫ ϩ ϩ III Me2N I ϩ 3 Me2N II 2 (solvent)(F8)Fe –(O2 )(3) [referred to subsequently as 2[(L )Cu ] (2) O2 [{(L )Cu }2(O2)] [3] III Ϫ (F8)Fe -(O2 )(3)], as elucidated from stopped-flow kinetic 3 2, studies. Superoxo species subsequently reacts with form-Ϫ I͞ ␮ III 2 presence of a nitrile strongly inhibits Cu O2 reactivity. With ing the heterobinuclear -peroxo complex [(F8)Fe -(O )– ϩ 2 II Me2N 4 this background, we have designed a solvent system, Cu (L )] ( ), which has been characterized by ultraviolet– ͞ ͞ visible (UV-Vis), nuclear magnetic resonance (NMR), and CH2Cl2 6% (vol vol) EtCN, that should force a reaction of resonance Raman (RR) spectroscopies. Notably, the tridentate an initially formed iron–superoxide species with our copper(I) versus tetradentate Cu chelation leads to striking differences complex, in close analogy to one of the key steps of the O2- in the nature of the peroxo moiety (i.e., the O–O stretching reduction cycle in cytochrome c oxidase (see above). In III 2Ϫ ͞ frequency) formed in 4 versus that in [(F8)Fe –(O2 )– CH2Cl2 6% EtCN, the EtCN (i) serves as an axial base heme CuII(TMPA)]ϩ (6). The ␮-peroxo complex 4 thermally trans- ligand promoting iron–superoxide formation (Eq. 1) (26) and ϩ ϩ ␮ III II Me2N Me2N I ͞ forms to the -oxo complex [(F8)Fe -(O)–Cu (L )] (5), (ii) causes the [(L )Cu ] (2) O2 reaction to become insig- ͞ ͞ ͞ whose x-ray structure is described, and found to be dramatically nificant.** Yet, for the 1 2 O2 mixture in CH2Cl2 6% EtCN, III II ϩ different from that found in [(F8)Fe –(O)-Cu (TMPA)] (7). an iron–superoxo͞copper reaction occurs, leading to the ␮ III 2Ϫ -peroxo heterobinuclear species [(F8)Fe –(O2 )– Materials and Methods ϩ CuII(LMe2N)] (4). Such heme-peroxo-copper species were not II II Compounds (F8)Fe (1) (17, 25), (F8)Fe -d8 (1-d8) (17), and observed from the reactions of 3 with analogous CuI com- Me2N I [(L )Cu ]B(C6F5)4 (2) (25) have been previously described. R I ϩ ϭ ␮ III 2Ϫ plexes ([(L )Cu ] , where L N,N-bis[2-(2-pyridyl)ethyl]- The dioxygen adduct, -peroxo complex [(F8)Fe –(O )– ϩ 2 methylamine and R ϭ 4-pyridyl substituents Cl, H, or OMe); CuII(LMe2N)] (4), was generated by bubbling O through 2 the electron-donating dimethylamino groups in the LMe2N in- 178–193 K solutions of 1 and 2 that were prepared in an inert I 3 4 atmosphere glove box. The ␮-oxo complex 5 was isolated by crease reactivity of Cu with leading to formation of . ͞ Benchtop UV-Vis spectroscopic changes of the oxygena- generation of 4 in CH2Cl2 10% CH3CN with subsequent ther- II tion reaction of an equimolar mixture of (F8)Fe (1) and mal transformation (193 K to room temperature, 1 h) to the Me N I ϩ product. Precipitation with heptane gave microcrystalline [(L 2 )Cu ] (2) are shown in Fig. 2. The reduced mixture has III II Me2N ϩ ͞ [(F8)Fe –(O)–Cu (L )] (5) in 58% yield. See Experimen- absorptions at 415, 425, and 527 nm at 193 K in CH2Cl2 6% tal Methods in Supporting Text, which is published as support- EtCN solution. Bubbling with dioxygen produces a stable dioxy- ing information on the PNAS web site, www.pnas.org, for fur- gen adduct with new spectral features at 420, 540, and 567 nm. We formulate this as a peroxo complex (probably as two iso- ther details concerning syntheses and spectroscopic methods. Ϫ III 2 II Me2N ϩ meric forms), [(F8)Fe –(O2 )–Cu (L )] (4), based on RR Results and Discussion and 1H and 2H NMR spectroscopies (see below). Complex 4 is ͞ II ͞ Me2N I ؉ (F8)Fe (1) [(L )Cu ] (2) O2 Reaction: Benchtop UV-Vis Spectros- not stable at higher temperatures (Ն203 K), primarily leading to II copy. Investigations of the independent chemistry for (F8)Fe ␮ ϩ the formation of -oxo complex 5, with its characteristic red- ͞ Me2N I ͞ (1) O2 (26) and [(L )Cu ] (2) O2 (24) have been de- shifted Soret band (449 nm) and broad 558-nm absorption. scribed. The O2 adduct observed from low-temperature reac- Trace amounts of water, always present in the solvent, result in ͞ ϭ tions of 1 with O2 is solvent dependent (Fe O2 1:1 or 2:1). some hydrolysis of the ␮-oxo bridge, especially in the low com- In coordinating solvents [e.g., tetrahydrofuran (THF) and plex concentrations (Ϸ10Ϫ5 M) of the UV-Vis experiment. Thus, 1 ϩ propionitrile (EtCN)], oxygenation of complex yields a mixtures of [(F )FeIII–O–CuII(LMe2N)] (5) (449 and 558 nm) 1 8 heme-superoxo complex (Eq. ), whereas only the peroxo- and (F )FeIII–OH (407 and 571 nm) (26) are observed, as shown bridged homobinuclear diiron complex is observed in nonco- 8 Me2N I ϩ in Fig. 2. ordinating solvents (CH2Cl2, toluene; Eq. 2). [(L )Cu ] ␮ (2) reacts with O2, yielding a bis- -oxo dicopper(III) and side-on peroxo dicopper(II) equilibrium mixture at low tem- NMR Spectroscopy. The oxygenation reaction of an equimolar II Me2N I ϩ perature (Eq. 3) (24), yet we observe that the mixture of (F8)Fe (1) and [(L )Cu ] (2) was followed by low-temperature NMR spectroscopy. To unambiguously identify pyrrole resonances, which aid iron oxidation and spin-state as- 2 II ϩ |L; IIIϪ Ϫ signments (27), complementary H NMR spectroscopic investi- (F8)Fe (1) O2 (solvent)Fe (O2 ) [1]

͞ ͞ ͞ ͞ II ϩ |L; III 2Ϫ **A minor 2 O2 reaction is observed in CH2Cl2 6% EtCN, but it does not affect the 1 2 O2 2(F8)Fe (1) O2 [(F8)Fe ]2–(O2 ) [2] kinetics described in this report.

3624 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0737180100 Kim et al. Downloaded by guest on September 26, 2021 SPECIAL FEATURE

1 II Me N I ϩ Fig. 3. H NMR spectra of the oxygenation reaction of (F8)Fe (1)͞[(L 2 )Cu ] III 2Ϫ II Me N ϩ Fig. 4. RR spectra of [(F8)Fe –(O )–Cu (L 2 )] (4) formed by oxygenation ͞ III 2 (2)inCD2Cl2 6% EtCN-d5 at 178 K. (A) Equimolar mixture of 1 and 2.(B) [(F8)Fe – 16 16 ͞18 16 16 18 Ϫ with O2 (trace A), a scrambled O O gas containing 25% O2, 50% O O, 2 II Me2N ϩ (O2 )–Cu (L )] (4) (red). (C) A product mixture obtained after warming 4, 18 18 and 25% O2 (trace B), or O2 (trace C). The difference spectrum of A minus III II Me2N ϩ III containing [(F8)Fe –O–Cu (L )] (5) (green) and (F8)Fe –OH (pink). C is shown as trace D. All spectra were obtained at Ϸ90 K with excitation at 413 nm. gations were also carried out employing a deuterated (␤-pyrrolic ͞ ϷϪ Ϫ1 ␯ 16 18 hydrogens) analog of 1 (1-d8). In CD2Cl2 6% EtCN-d5 at 178 K, isotope shifts of 20 cm for as( O– O) vibrations. Large 1H NMR spectra of the reduced 1͞2 mixture exhibit only dia- porphyrin vibrations hinder direct visualization of additional 16 18 magnetic resonances (i.e., 1–10 ppm, Fig. 3A), indicating the bands originating from O– O peroxo species in Fig. 4, but CHEMISTRY presence of a low-spin six-coordinate ferrous heme (d6,Sϭ 0; their presence becomes apparent in difference spectra (see Fig. including two EtCN axial base ligands) and a CuI complex (d10, 8, which is published as supporting information on the PNAS ϭ S 0). Bubbling dry O2 into a precooled NMR tube containing web site). In trace B (Fig. 4 trace B), the relative intensity of the Ϫ ϩ III 2 II Me2N Ϫ1 1 and 2 leads to the formation of [(F8)Fe –(O2 )–Cu (L )] isotope-sensitive band at 767 cm corresponds to 25% of that Ϸ 16 (4), with downfield-shifted pyrrole resonances at 110 ppm (Fig. observed in a sample formed with pure O2 gas (Fig. 4 trace 3B), verified by 2H NMR spectroscopy (Fig. 7, which is pub- A). Similarly, the intensity of the 707-cmϪ1 band obtained from lished as supporting information on the PNAS web site). This the scrambled gas samples represents 25% of the intensity ob- observed pyrrole chemical shift position excludes the possibility 18 Ϫ served with the pure O2 gas (Fig. 4 trace C). We can therefore III IV that 4 is (i)aniron–superoxide, i.e., (F8)Fe –(O )(3) (8.9 ppm, ␯ ␯ A 2 Ϫ assign these signals to peroxo (O–O), rather than a (Fe O) ␮ III 2 III II 193 K), (ii) the -peroxo complex [(F8)Fe ]2–(O2 ) (17.5 ppm, stretch or a ␯(Fe –O–Cu ), where the dioxygen bond has been IVA 193 K), (iii) the ferryl-oxo species (F8)Fe O (3.5 ppm, 193 cleaved and 50% intensity ratios are expected. III Ϸ K), or (iv)(F8)Fe –OH ( 135 ppm, 178 K) (26). The 110 ppm Assignment of the O–O stretching vibrations to a homo- Ϫ Me2N II 2 2ϩ pyrrole resonance in 4 is in the range for high-spin Fe(III), yet it nuclear peroxo–dicopper complex, [{(L )Cu }2(O2 )] , has a significantly diminished downfield shift compared with that was ruled out in a control experiment where the oxygenation ϭ ͞ III ϭ Ϫ Me N I ϩ seen for S 5 2 complexes such as (F8)Fe –X(X OH or reaction was carried out with a 5-fold excess of [(L 2 )Cu ] Ϫ Ϸ II Cl ; 135 ppm at 178 K). This observation is consistent with (2) over (F8)Fe (1). No significant difference could be ob- the presence of strong antiferromagnetic coupling between high- served in the RR spectra, thus affirming that the reaction of III 5 ϭ ͞ II 9 ϭ ͞ III spin Fe (d ,S 5 2) and Cu (d ,S 1 2) in [(F8)Fe – 2 with O2 is not of consequence in the presence of nitrile. Ϫ ϩ Ϫ 2 II Me2N Me2N II 2 2ϩ (O2 )–Cu (L )] (4). Cu-ligand hydrogen resonances are {Note: independent RR studies on [{(L )Cu }2(O2 )] observed here for the LMe2N moiety in 4 (25, Ϫ24, Ϫ32, and Ϫ46 formed in solvents with no EtCN present show a ␯(O–O) vi- Ϫ1 ⌬18 ϭϪ Ϫ1 ppm, Fig. 4B). The position of the pyrrole resonances and the bration at 729 cm , O2 40 cm , in acetone (31).} observable copper ligand hydrogen signals are characteristic fea- Ϫ While anionic mononuclear heme–peroxo species III II ϭ 2 2Ϫ ϭ III 2Ϫ Ϫ tures of (porphyrinate)Fe –X–Cu (X O2 or O )S 2 [(F8)Fe -(O2 )] might conceivably form, as is known with systems, as previously described (14, 15, 17, 28). other porphyrinate ligands, they display ␯(O–O) at significantly higher frequencies, i.e., Ͼ800 cmϪ1 (32, 33). Peroxo III 2Ϫ RR Spectroscopy. Soret excitation of [(F8)Fe –(O2 )– stretching vibrations from homonuclear diheme–peroxo com- ϩ Ϫ II Me2N III 2 ϭ Cu (L )] (4) at 413 nm results in RR spectra dominated by plexes [(P)Fe ]2-(O2 )(P porphyrinate) have not been strong porphyrin skeletal modes and weak iron-ligand vibrations observed with RR spectroscopy, but low-temperature NMR (29, 30). Porphyrin skeletal modes in the high-frequency region spectroscopy in any case rules out such an assignment (see of the RR spectra are consistent with a five-coordinate high-spin above). The RR results clearly establish that two very similar III 2Ϫ ␯ ferric heme species as previously observed in [(F8)Fe –(O2 )– iron-porphyrinate peroxo species with (O–O) at 767 and 752 ϩ Ϫ II Ϫ1 III 2 II Me2N ϩ Cu (TMPA)] (6) (14, 30). Comparison of low-temperature RR cm are present in [(F8)Fe –(O2 )–Cu (L )] (4) and ͞ 16 18 spectra of 4 generated in CH2Cl2 6% EtCN with O2 or O2 strongly suggest a heme–peroxo–copper complex that adopts Ϫ reveals two isotopic shifts of ՆϪ40 cm 1 that identify two bands two closely related isomer forms. These are not distinguish- at 767 and 752 cmϪ1 as putative ␯(O–O) peroxo stretching vibra- able by UV-Vis or NMR spectroscopy. tions (Fig. 4). The latter assignment was confirmed by comple- From the work of Collman, Naruta, and ourselves, there mentary experiments with scrambled isotope gas mixtures com- are now several examples of heme–peroxo–copper complexes 16 16 18 18 posed of 25% O2, 50% O O, and 25% O2. Differential RR that are characterized by RR spectroscopy (Table 1). Our 16 spectra, i.e., compared with spectra with pure O2, are expected previous investigations of two closely related systems, ‘‘un- III 2Ϫ II ϩ to present additional intensity at intermediate frequency with tethered’’ [(F8)Fe –(O2 )–Cu (TMPA)] (6) and an ana-

Kim et al. PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 3625 Downloaded by guest on September 26, 2021 Table 1. RR data of heme–peroxo–Cu complexes

16 18 Ϫ1 Complex ␯( O2)(⌬( O2)), cm Ref.

Ϫ III 2 II Me2N ϩ [(F8)Fe –(O2 )–Cu (L )] (4) 767 (41), 752 (45) This work III 2Ϫ II ϩ [(F8)Fe –(O2 )–Cu (TMPA)] (6) 808 (46) 14 6 III 2Ϫ II ϩ [( L)Fe –(O2 )–Cu ] 788 (44) 15 III II 2Ϫ ϩ [(PTPA)Fe Cu –(O2 )] 803 (44) 37 III 2Ϫ II ϩ [(P5-MeTPA)Fe –(O2 )–Cu ] 793 (42) 37 III 2Ϫ II ϩ [(PTACN)Fe –(O2 )–Cu ] 758 (18) 38

PTPA, tris(2-picolylamine)- or P5-MeTPA, tris(2-(5methyl)pyridylmethyl)amine- linked tetraphenylporphyrinate; PTACN, triazacyclononane-capped tetraaryl- porphyrin.

logue where the TMPA moiety is covalently attached to the 6 III 2Ϫ II ϩ heme, ‘‘tethered’’ [( L)Fe –(O2 )–Cu ] , show a significant 20-cmϪ1 difference in ␯(O–O) stretching vibrations (Table 1). This observation suggests that ligand constraints in the tethered complexes can strongly influence peroxo O–O stretching vibrations. Systems with nonheme diferric ␮-1,2 peroxo groups, where differences in bond angles influence coupling of ␯(O–O) with ␯(Fe–O) vibrations, lead to large variations in ␯ II Ϫ4 Me N I ϩ Ϫ4 observed frequencies (34–36). The differences in (O–O) ob- Fig. 5. (A) Reaction of (F8)Fe (1) (3.43 ⅐ 10 M) and [(L 2 )Cu ] (2) (3.69 ⅐ 10 Ϫ3 served in this series of heme–peroxo–copper complexes (i.e., M) with O2 (2.20 ⅐ 10 M) at 168 K in CH2Cl2͞6% EtCN (50 out of a total of 172 Ϫ 6 III 2 II ϩ spectra shown). (Inset) Absorbance–time traces at 541 nm [formation and decay 6 vs. [( L)Fe –(O2 )–Cu ] and 4) likely relate to changes in III Ϫ Ϸ ⅐ Ϫ4 ⅐ Ϫ3 Fe–O–O angles and possibly a transition from ␮-1,2 to of (F8)Fe –(O2 )(3) over 3.5 s]. (B) Reaction of 1 (2.67 10 M) with O2 (2.20 10 ͞ ␮-␩2:␩2 bridging geometry (see below). In rather similar com- M) at 168 K in CH2Cl2 6% EtCN (50 out of a total of 150 spectra shown). (Inset) Absorbance–time trace at 541 nm (over Ϸ1.7 s) for formation of 3. plexes, Naruta and coworkers (37) reported that the incorporation of electron-donating copper-ligand substituents results III II 2Ϫ ϩ in a weaker O–O bond. (Compare [(PTPA)Fe Cu –(O2 )] The observation (from RR spectroscopy) of two isomers in III II 2Ϫ ϩ Ϫ III 2 II Me2N ϩ and [(P5-MeTPA)Fe Cu –(O2 )] ; Table 1.) [(F8)Fe –(O2 )–Cu (L )] (4) may be a result of differ- The present studies reveal that tridentate versus tetradentate ent conformers caused by different copper ligand orienta- copper chelation plays a key role in determining heme–peroxo– tions. If the copper center in 4 has a square-pyramidal geom- copper complex stability and peroxo ␯(O–O) stretching vibra- etry, as is well known for five-coordinate complexes Ϫ ϩ III 2 II Me2N R ϭ tion. [(F8)Fe –(O2 )–Cu (L )] (4) is not as thermally sta- containing L (R H, MeO, or Me2N) (43), there are two ble as the tetradentate containing compounds. For example, reasonable possibilities for the positioning of the LMe2N ligan- Ϫ Ϫ Ϸ III 2 III 2 II Me2N ϩ 4 decomposes above 213 K, whereas [(F8)Fe –(O2 )– d–copper complex in [(F8)Fe –(O2 )–Cu (L )] (4)in ϩ CuII(TMPA)] (6) has room temperature stability in MeCN so- the basal plane: (i) one pyridyl and one alkylamino group are lution. From a more quantitative perspective, the RR studies trans to the O atoms of the peroxo bridging ligand or (ii)two (see above) show that complex 4 exhibits significantly lower pyridyl groups are trans to the O atoms. values in ␯(O–O) vibrations (752 and 767 cmϪ1) than does 6 ͞ Ϫ1 Ϫ1 II ͞ Me2N I ؉ (808 cm ), a decrease in frequency of Ն40 cm . The ␯(O–O) Kinetics of the Reaction of (F8)Fe (1) [(L )Cu ] (2) O2 and Ϫ1 ͞ vibrations in 4 are comparable with the 758-cm value (Table 1 O2. The reaction of 1 and 2 with O2 was investigated by III II 2Ϫ ϩ ͞ 1) seen by Collman et al. (38) in the [(PTACN)Fe Cu –(O2 )] stopped-flow kinetics in CH2Cl2 6% EtCN. Fig. 5A shows spec- complex, where a tridentate copper ligand triazacyclononane troscopic monitoring in the heme ␣͞␤ region (470 nm to 625 (TACN) as well as heme imidazole axial base ligand was used. nm) over the time period of 3.5 s. A band at 531 nm (reduced The origin of the instability and O–O bond weakening are not complex 1) decays and an intermediate with 541-nm absorption, III Ϫ yet fully resolved, but in comparison to the tetradentate Cu liga- ascribed to (F8)Fe –(O2 )(3) (see below), grows in during the III 2Ϫ II ϩ first Ϸ0.25 s (Fig. 5A). This then decays over 2–3stomorecom- tion in [(F8)Fe –(O2 )–Cu (TMPA)] (6) or other complexes, Ϫ ϩ Ϸ Ϸ III 2 II Me2N plex spectra with broad maxima at 540 nm and 565 nm, tridentate Cu ligation in [(F8)Fe –(O )–Cu (L )] (4) pro- 2 ␮ III 2Ϫ which we ascribe to the -peroxo species [(F8)Fe –(O2 )– vides an additional open coordination site. As is now well known ϩ II Me2N ␭ in copper(I)͞dioxygen chemistry, this could result in ␮-␩2:␩2 Cu (L )] (4). (Small differences in max values seen in the side-on versus ␮-1,2-peroxo end-on ligation, which consider- kinetics studies here, compared with benchtop UV-Vis monitor- ably reduces the ␯(O–O) value (805–830 cmϪ1 going down to ing, are ascribed to the use of different spectrometers at differ- Ϫ1 ent temperatures.) 710–760 cm ) (20, 39, 40) and poises the Cu2(O2) peroxo- Fig. 5B shows the UV-Vis spectra for the oxygenation of a dicopper(II) moiety for O–O bond cleavage (41). Thus, along ͞ solution containing only 1 in CH2Cl2 6% EtCN. As in the with the knowledge that side-on (as well as end-on) (42) liga- ϩ II 1 ͞ Me2N I 2 1 tion for heme-peroxo species exists, we suggest that a side-on (F8)Fe ( ) [(L )Cu ] ( ) mixture, has an absorbance maximum near 531 nm, which rapidly decreases upon reaction heme-peroxo-copper intermediate structure (left structure in III Ϫ with dioxygen. Concomitant generation of (F8)Fe –(O2 )(3) diagram below) is a strong possibility for 4. ␭ ϭ ( max 541 nm) occurs and its formation is complete within Ϸ0.5 s (Fig. 5B Inset). This species is quite stable at low temperatures. RR characterization of 3 formed in THF unambiguously confirms this to be a low-spin six-coordinate superoxo ␯ ϭ Ϫ1 ⌬ 18 ϭϪ Ϫ1 species, with (O–O) 1178 cm [ ( O2) 64 cm ] ␯ ϭ Ϫ1 ⌬ 18 ϭϪ Ϫ1 and (Fe–O) 568 cm [ ( O2) 24 cm ] (see Fig. 9, which is published as supporting information on the PNAS

3626 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0737180100 Kim et al. Downloaded by guest on September 26, 2021 III ؊ Table 2. Kinetic parameters for the formation of (F8)Fe –(O2 ) (3)

II Me N I ϩ II 2 SPECIAL FEATURE (F8)Fe (1)͞[(L )Cu ] (2) ϩ O2 (F8)Fe (1) ϩ O2

† † § Parameter CH2Cl2͞6% EtCN* THF͞6% EtCN* CH2Cl2͞6% EtCN* THF͞6% EtCN* EtCN

⌬H‡,kJ͞mol NA 20 Ϯ 1 41.2 Ϯ 0.6 21.0 Ϯ 0.6 38.6 Ϯ 0.4 ⌬S‡,J͞mol⅐KNAϪ25 Ϯ 674Ϯ 4 Ϫ19 Ϯ 442Ϯ 2 Ϫ1 Ϫ1 3 4 3 5 2 k1,M ⅐s 168 K (6 Ϯ 2) ⅐ 10 (8.5 Ϯ 0.2) ⅐ 10 (4.00 Ϯ 0.16) ⅐ 10 (1.23 Ϯ 0.04) ⅐ 10 (5.5 Ϯ 0.2) ⅐ 10 Ϫ1 Ϫ1 4 5 4 5 3 k1,M ⅐s 183 K (6 Ϯ 2) ⅐ 10 (3.05 Ϯ 0.14) ⅐ 10 (4.86 Ϯ 0.10) ⅐ 10 (4.21 Ϯ 0.08) ⅐ 10 (5.57 Ϯ 0.04) ⅐ 10 Ϫ1 Ϫ1 5 5 5 6 4 k1,M ⅐s 198 K (5 Ϯ 1) ⅐ 10 (9.0 Ϯ 0.8) ⅐ 10 (4.07 Ϯ 0.16) ⅐ 10 (1.29 Ϯ 0.06) ⅐ 10 (4.19 Ϯ 0.06) ⅐ 10

Data are presented as best estimates with twice their standard errors. NA, not applicable. Sources are as follows: *, this work; †, preliminary data; §, ref. 26.

web site), in line with previous chemical and other spectro- that this occurs by a disproportionation reaction, i.e., Ϫ ϩ III 2 II Me2N 3 III scopic studies (26). 2[(F8)Fe –(O2 )–Cu (L )] (4) 2[(F8)Fe –O– II II Me2N ϩ The data obtained for the reaction of (F8)Fe (1) with O2 Cu (L )]B(C6F5)4 (5) O2. The product 5 is highly mois- ϩ II ͞ Me2N I as well as for (F8)Fe (1) [(L )Cu ] (2) with O2 in ture sensitive, but is stable at room temperature under inert ͞ ͞ CH2Cl2 6% EtCN and in THF 6% EtCN are presented in atmosphere. Complex 5 (Fig. 6) possesses an oxide bridge Table 2. They allow us to compare formation of (F )FeIII– between a square-pyramidal iron(III) porphyrinate and a Ϫ 8 (O2 )(3) in the presence or absence of copper. In fact, the highly distorted tetracoordinate copper(II) ion. Notably, results show that copper complex 2 has essentially no effect aFeIII–(X)–CuII moiety, with three copper–N donors and a ϭ Ϫ Ϫ on the formation of 3 as seen from the rate constants and bridging ligand (X O2 ,OH ,orH2O) is observed in the activation parameters obtained (Table 2). active-site structure of the ba3-type cytochrome c oxidase (5). II The kinetics of formation of (F8)Fe (1) in the various sol- The Fe–N bond distances in 5 and the large displacement of vents used indicate a high degree of similarity, but there are the iron atom from the porphyrinate ligand (0.55 Å) are very certain differences in detail. Below 200 K, the formation of similar to those of five-coordinate high-spin Fe(III) porphyri- III Ϫ ͞ (F8)Fe –(O2 )(3)inTHF 6% EtCN is faster compared with nates (44). The Cu–N bond distances are typical for Cu(II)

͞ CHEMISTRY the same reaction in CH2Cl2 6% EtCN, primarily because of with alkyl and͞or pyridylamine ligation (Fig. 6). Both pyridyl an effect of activation enthalpy (Table 2). Because in groups on the copper ligand are placed ‘‘above’’ and between ͞ CH2Cl2 6% EtCN, EtCN binds to 1 as an axial base ligand the four difluorophenyl meso-substituents of the heme (low-spin six- coordinate; see above), the enhanced rate of O2 (Fig. 6). ͞ III reaction with 1 in THF 6% EtCN therefore must be due to The most significant structural feature of [(F8)Fe –O– ϩ an effect of THF. Factors such as THF versus EtCN binding CuII(LMe2N)] (5) is the Fe–O–Cu core. Table 3 shows compari- strengths, lability, electron-donating ability, and iron(II) spin sons of the metal–oxygen bond distances and the (Fe–O–Cu) ͞ III II state need to be considered. We note that in CH2Cl2 6% bond angle for 5 with other (porphyrinate)Fe –O–Cu com- EtCN, ⌬S‡ ϭ 74 Ϯ 4J͞mol⅐K is suggestive of dissociative loss plexes. The metal–oxygen bond distances are all similar and ͞ of EtCN during O2 binding, whereas in THF 6% EtCN a characteristically short, as has been described previously (45). negative value (⌬S‡ ϭϪ19 Ϯ 4J͞mol⅐K) perhaps indicates The Fe–O bond length in 5 is similar to those of ␮-oxo-diiron- II an associative binding of O2 to a pentacoordinate (THF)Fe (porphyrinates) (48), and the short Cu–O bond distance species. (Note that in THF, 1 is high-spin at all tempera- [1.852(2) Å] is not far off from values observed for bis-␮-oxodi- III Ϫ tures.) Comparing formation of (F8)Fe –(O2 )(3) in EtCN copper(III) complexes (Cu–O Ϸ 1.8 Å) (21). Most notably, ͞ and CH2Cl2 6% EtCN, the activation enthalpies are nearly there is a large difference in the (Fe–O–Cu) bond angle in 5 identical (Table 2); the activation entropy varies because the compared with complexes with a similar chemical framework. II III 2Ϫ II equilibrium for binding of EtCN to (F8)Fe (1) lies further to Almost all (porphyrinate)Fe –(O )–Cu complexes with tetra- the right in pure EtCN. dentate copper ligands have near-linear core structures, but the ͞ ͞ In both CH2Cl2 6% EtCN and THF 6% EtCN, the decay Fe–O–Cu bridge in 5 is severely bent to an angle of 143.4° (Ta- III Ϫ of the band at 541 nm shows that (F8)Fe –(O2 )(3) is short- ble 3). A similar small angle (Ϸ140 Ϯ 3°) was deduced from an lived in the presence of copper complex 2, because of further x-ray absorption study on [(5L)Fe–(O)–Cu]ϩ, where tethering a ␮ III 2Ϫ reaction to give -peroxo species [(F8)Fe –(O2 )– TMPA chelate to the porphyrin imposes severe ligand con- ϩ CuII(LMe2N)] (4). As mentioned above, the reaction of ϩ Me2N I ͞ [(L )Cu ] (2) with O2 in CH2Cl2 6% EtCN is inconse- quential, but 2 does react with 3, i.e., a heme-bound O2 spe- ͞ cies. In CH2Cl2 6% EtCN (Fig. 5A), the half-life for decay of ϭ ϭ ⅐ Ϫ4 3 to form 4 at 168 K is t1/2 0.40 s ([Fe] 3.43 10 M, ϭ ⅐ Ϫ4 ϭ ⅐ Ϫ3 [Cu] 3.69 10 M, [O2] 2.21 10 M). A preliminary data analysis reveals that this transformation contains at least two relaxations. Because the RR data for solutions of 4 suggest the presence of two ␮-peroxo species (see above), paral- lel reactions (leading to two different products) have to be considered as a mechanistic possibility. For a full understand- ͞ ͞ ␮ ing of the reaction of 1 2 O2, including the decay of - III II Me2N ϩ peroxo complex 4 to [(F8)Fe –(O)–Cu (L )] (5), a complete kinetic analysis is required and is forthcoming.

III II Me2N ؉ X-Ray Structure and Properties of [(F8)Fe –O–Cu (L )] (5). III Fig. 6. ORTEP representation showing the cationic portion of [(F8)Fe –O– ␮ II Me N Warming the -peroxo complex 4 to room temperature Cu (L 2 )]B(C6F5)4 (5). Fe⅐⅐⅐Cu, 3.417 Å; Cu1–O1–Fe1, 143.44(12)°. See Tables leads to formation of 5; by analogy to the process shown to 4–8 and an expanded version of this ﬁgure with further details, which are occur with the TMPA ligand–Cu system (14), it is presumed published as supporting information on the PNAS web site.

Kim et al. PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 3627 Downloaded by guest on September 26, 2021 ϩ Table 3. Comparison of the core structures of for copper(I), in complex [(LMe2N)CuI] (2), whose reaction III 2؊ II ؉ II [(P)Fe –(O )–Cu ] complexes with (F8)Fe (1) and O2 leads first to the superoxo-iron(III) III Ϫ Distance, Å Angle, ° complex (F8)Fe –(O2 )(3), with subsequent formation of the Ϫ ϩ ␮ III 2 II Me2N -peroxo species [(F8)Fe –(O2 )–Cu (L )] (4). In a Complex Fe–OCu–OFe–O–Cu Ref. ␮ III slower reaction, 4 transforms to a -oxo complex [(F8)Fe – II Me N ϩ 5 1.747 (2) 1.852 (2) 143.4 (2) This work (O)–Cu (L 2 )] (5). From studies in a number of solvents 7 1.740 (5) 1.856 (5) 178.2 (4) 45 and solvent mixtures, we find that low temperatures (i.e., [(6L)Fe–O–Cu]ϩ 1.750 (4) 1.848 (4) 171.1 (3) 16 Ͻ205 K) are required to observe the fast formation of 3. Re- Ј ϩ [(OEP)Fe–O–Cu(tren )] 1.747 (6) 1.827 (6) 176.0 (7) 46 action rates are enhanced by the presence of THF, and the (F8)Fe–O–Fe(F8) 1.760 (2) 178.5 (8) 45 ϩ kinetics of formation of 3 are independent of the presence of [(5L)Fe–O–Cu] * 1.76 1.84 141 Ϯ 647 Ϫ III 2 II Me2N ϩ copper complex 2. [(F8)Fe –(O2 )–Cu (L )] (4) has OEP, dianion of octaethylporphyrin; trenЈ, tris[(N,N-dimethylamino)- characteristic UV-Vis and 1H NMR spectroscopic features, ethyl]amine. III 2Ϫ II which compare well with other Fe –O2 –Cu species em- *X-ray absorption spectroscopic data. ploying tetradentate Cu ligands; they are all S ϭ 2 systems with strong magnetic coupling between the iron and copper straints affecting structural, chemical, and physical centers. RR spectroscopy clearly identifies 4 as having a per- properties (47). oxo moiety; actually two such species are present in solution. Despite the difference in the Fe–O–Cu core structures, The strongly reduced ␯(O–O) values observed are ascribed to there is a strong magnetic coupling between iron (d5,Sϭ the tridentate Cu–LMe2N ligation; A ␮-␩2:␩2 side-on peroxo ͞ 9 ϭ ͞ III 5 2) and copper (d ,S 1 2) in both [(F8)Fe –O– III ϩ ϩ structure for 4 is a reasonable possibility. Complex [(F8)Fe – II Me2N ϩ Cu (L )] (5) and [(F8)Fe–O–Cu(TMPA)] (7). A low- II Me2N 5 ϭ 1 (O)–Cu (L )] ( ) is also a strongly coupled S 2 sys- temperature H NMR spectrum of 5 (Fig. 3C) exhibits an up- tem. The x-ray structure of 5 reveals a severely bent FeIII- field and downfield pattern of signals similar to the ␮-peroxo Ϫ O2 -CuII core, in contrast to near-linear analogs that possess species 4 (Fig. 3B). [A fully assigned room-temperature spectrum of 5 (Fig. 10) and a Curie plot (␦ vs. 1͞T; Fig. 11) are tetradentate copper chelates. This study adds significantly to published as supporting information on the PNAS web site.] ongoing investigations focused on how heme-copper centers Thus, 5 is also an S ϭ 2 spin system, supported by solution react with dioxygen, with particular consideration of the im- ␮ ϭ Ϯ Evans method magnetic moment determination ( eff 4.9 portant influence of the denticity (tetra- vs. tridentate) of the ␮ 0.2 B at room temperature). Strong magnetic coupling is a copper ligand. general feature of ␮-oxo dimetal complexes, over a wide range of M–O–MЈ angles (48). This work was supported by National Institutes of Health Grants GM60353 (to K.D.K.), GM18865 (to P.M.-L.) and GM20805 (to Summary͞Conclusions. In continuing studies of heme-copper͞ M.E.H.), and financial support from the Swiss National Science dioxygen chemistry, we have used a novel tridentate ligand Foundation (to A.D.Z.).

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