Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands.

In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O2 is reduced to H2O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and ∼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.

Oxygen Reduction by Iron Porphyrins with Covalently Attached Pendent Phenol and Quinol.

Phenols and quinols participate in both proton transfer and electron transfer processes in nature either in distinct elementary steps or in a concerted fashion. Recent investigations using synthetic heme/Cu models and iron porphyrins have indicated that phenols/quinols can react with both ferric superoxide and ferric peroxide intermediates formed during O2 reduction through a proton coupled electron transfer (PCET) process as well as via hydrogen atom transfer (HAT). Oxygen reduction by iron porphyrins bearing covalently attached pendant phenol and quinol groups is investigated. The data show that both of these can electrochemically reduce O2 selectively by 4e-/4H+ to H2O with very similar rates. However, the mechanism of the reaction, investigated both using heterogeneous electrochemistry and by trapping intermediates in organic solutions, can be either PCET or HAT and is governed by the thermodynamics of these intermediates involved. The results suggest that, while the reduction of the FeIII-O2̇- species to FeIII-OOH proceeds via PCET when a pendant phenol is present, it follows a HAT pathway with a pendant quinol. In the absence of the hydroxyl group the O2 reduction proceeds via an electron transfer followed by proton transfer to the FeIII-O2̇- species. The hydrogen bonding from the pendant phenol group to FeIII-O2̇- and FeIII-OOH species provides a unique advantage to the PCET process by lowering the inner-sphere reorganization energy by limiting the elongation of the O-O bond upon reduction.

Resonance Raman Spectroscopy and Density Functional Theory Calculations on Ferrous Porphyrin Dioxygen Adducts with Different Axial Ligands: Correlation of Ground State Wave Function and Geometric Parameters with Experimental Vibrational Frequencies.

A dioxygen adduct of ferrous porphyrin is an important chemical species in nature as it is a common intermediate in all oxygen transfer, storage, reducing, and activating heme enzymes. The ground state (GS) wave function of this complex has been investigated using several techniques like resonance Raman (rR), Mossbauer, and X-ray absorption spectroscopies. The Fe-O and O-O vibrations of these six-coordinated diamagnetic species show a positive correlation with each other in contrast to analogous ferrous carbonyl complexes where the Fe-CO vibration correlates negatively with the C-O vibration due to a synergistic effect. In this Article, three Fe-porphyrins with different axial ligands (imidazole, phenolate, and thiolate) are investigated using rR spectroscopy and density functional theory (DFT) calculations. The GS wave functions of these species are analyzed, and the contribution of the three primary bonding interactions in the Fe-O2 unit (a σ interaction from the in-plane π* of the superoxide to the vacant dz2 of Fe, a π donation from the out of plane (oop) π* to the dπ orbital of Fe, and a π back bonding interaction from the dπ orbital of Fe to the oop π* of the superoxide) to the calculated Fe-O and O-O vibrations and bond lengths are deconvoluted using a MO theory framework. The GS wave function provides a basis for the correlations observed between different vibrational and geometric parameters of these dioxygen adducts. Furthermore, the correlations obtained allows estimation of the GS wave function and geometry of these species (both natural and artificial) using their experimentally observed Fe-O/O-O vibrations. The wave functions thus extracted from the experimental vibrational data reported offer insight into the role of the axial ligand and hydrogen bonding on the geometric and electronic structures of these crucial chemical species in different protein active sites.

Hydrogen atom abstraction by synthetic heme ferric superoxide and hydroperoxide species.

To date, artificial dioxygen adducts of heme have not been demonstrated to be able to oxidize organic substrates in sharp contrast to their non-heme analogues and naturally occurring enzymes like heme dioxygenases. To address this apparent anomaly, an iron porphyrin complex is synthesized which includes a pendant quinol group. The corresponding dioxygen bound iron porphyrin species is demonstrated to perform hydrogen atom transfer (HAT) from the quinol group appended to the porphyrin ligand. The resultant ferric peroxide, formed by the first HAT, performs a 2nd HAT generating a ferryl species (FeIV[double bond, length as m-dash]O) and resulting in the 2e-/2H+ oxidation of the pendant hydroquinone to quinone.

Effect of hydrogen bonding on innocent and non-innocent axial ligands bound to iron porphyrins.

Most known heme enzymes utilize hydrogen bonding interactions in their active sites to control electronic and geometric structures and the ensuing reactivity. The details of these weak 2nd sphere interactions are slowly unravelling through spectroscopic and theoretical investigations in addition to biochemical studies. In synthetic Fe porphyrins, nature of hydrogen bonding by iron bound hydroxide ligand (H bond acceptor or donor) is found to alter the spin state of Fe in a series of iron hydroxide complexes. In this study, a series of Fe porphyrins having a triazole ring appended in the distal site of the porphyrin macrocycle were synthesized. The triazole rings were substituted to systematically alter their electron densities, which tune their H bonding to water molecules trapped inside the distal cavity, and in turn H bonds to the axial ligands bound to the iron porphyrin. Resonance Raman data indicated that the metal ligand bond strength changes with the change in substituents on triazole rings for innocent ligands like hydroxide, as well as non-innocent ligands like oxygen, albeit the mechanisms by which hydrogen bonding affects these are very different. Additionally, H bonding interaction was also found to alter the pKa of ferric hydroxide complexes.

Interplay of Electronic Cooperativity and Exchange Coupling in Regulating the Reactivity of Diiron(IV)-oxo Complexes towards C-H and O-H Bond Activation.

Activation of inert C-H bonds such as those of methane are extremely challenging for chemists but in nature, the soluble methane monooxygenase (sMMO) enzyme readily oxidizes methane to methanol by using a diiron(IV) species. This has prompted chemists to look for similar model systems. Recently, a (µ-oxo)bis(µ-carboxamido)diiron(IV) ([FeIV2 O(L)2 ]2+ L=N,N-bis-(3',5'-dimethyl-4'-methoxypyridyl-2'-methyl)-N'-acetyl-1,2-diaminoethane) complex has been generated by bulk electrolysis and this species activates inert C-H bonds almost 1000 times faster than mononuclear FeIV =O species and at the same time selectively activates O-H bonds of alcohols. The very high reactivity and selectivity of this species is puzzling and herein we use extensive DFT calculations to shed light on this aspect. We have studied the electronic and spectral features of diiron {FeIII -µ(O)-FeIII }+2 (complex I), {FeIII -µ(O)-FeIV }+3 (II), and {FeIV -µ(O)-FeIV }+4 (III) complexes. Strong antiferromagnetic coupling between the Fe centers leads to spin-coupled S=0, S=3/2, and S=0 ground state for species I-III respectively. The mechanistic study of the C-H and O-H bond activation reveals a multistate reactivity scenario where C-H bond activation is found to occur through the S=4 spin-coupled state corresponding to the high-spin state of individual FeIV centers. The O-H bond activation on the other hand, occurs through the S=2 spin-coupled state corresponding to an intermediate state of individual FeIV centers. Molecular orbital analysis reveals σ-π/π-π channels for the reactivity. The nature of the magnetic exchange interaction is found to be switched during the course of the reaction and this offers lower energy pathways. Significant electronic cooperativity between two metal centers during the course of the reaction has been witnessed and this uncovers the reason behind the efficiency and selectivity observed. The catalyst is found to prudently choose the desired spin states based on the nature of the substrate to effect the catalytic transformations. These findings suggest that the presence of such factors play a role in the reactivity of dinuclear metalloenzymes such as sMMO.

Mechanism of Reduction of Ferric Porphyrins by Sulfide: Identification of a Low Spin FeIII-SH Intermediate.

The reaction of FeIII porphyrin complexes bearing distal hydrogen bonding residues with sulfide/hydrosulfide is kinetically monitored to reveal the presence of an intermediate and a kH/kD of 3.0. This intermediate is trapped at low temperatures and investigated with resonance Raman and electron paramagnetic resonance spectroscopy. The results, corroborated by density functional theory calculations, indicate that this species is a six-coordinate low spin hydrosulfide bound ferric porphyrin. The homolytic cleavage of the FeIII-SH bond resulting in the formation of a ferrous porphyrin and hydrosulfide radical (trapped with 5,5-dimethyl-1-pyrrilone-N-oxide) is found to be the overall rate-determining step of the reaction.

Iron porphyrins with a hydrogen bonding cavity: effect of weak interactions on their electronic structure and reactivity.

The electronic structure and reactivity of iron porphyrin complexes bearing 2nd sphere hydrogen bonding residues have been investigated over the last few years. The presence of these weak interactions alters the spin ground state, and axial ligand bonding and provides a proton translocation pathway into the active site. Mechanistic investigations in organic as well as aqueous media demonstrate how controlled delivery of protons is fundamental in dictating the selectivity of a multi-electron multi-proton process like the reduction of dioxygen to water.

Second sphere control of spin state: Differential tuning of axial ligand bonds in ferric porphyrin complexes by hydrogen bonding.

An iron porphyrin with a pre-organized hydrogen bonding (H-Bonding) distal architecture is utilized to avoid the inherent loss of entropy associated with H-Bonding from solvent (water) and mimic the behavior of metallo-enzyme active sites attributed to H-Bonding interactions of active site with the 2nd sphere residues. Resonance Raman (rR) data on these iron porphyrin complexes indicate that H-Bonding to an axial ligand like hydroxide can result in both stronger or weaker Fe(III)-OH bond relative to iron porphyrin complexes. The 6-coordinate (6C) complexes bearing water derived axial ligands, trans to imidazole or thiolate axial ligand with H-Bonding stabilize a low spin (LS) ground state (GS) when a complex without H-Bonding stabilizes a high spin (HS) ground state. DFT calculations reproduce the trend in the experimental data and provide a mechanism of how H-Bonding can indeed lead to stronger metal ligand bonds when the axial ligand donates an H-Bond and lead to weaker metal ligand bonds when the axial ligand accepts an H-Bond. The experimental and computational results explain how a weak Fe(III)-OH bond (due to H-Bonding) can lead to the stabilization of low spin ground state in synthetic mimics and in enzymes containing iron porphyrin active sites. H-Bonding to a water ligand bound to a reduced ferrous active site can only strengthen the Fe(II)-OH2 bond and thus exclusion of water and hydrophilic residues from distal sites of O2 binding/activating heme proteins is necessary to avoid inhibition of O2 binding by water. These results help demonstrate the predominant role played by H-Bonding and subtle changes in its orientation in determining the geometric and electronic structure of iron porphyrin based active sites in nature.