Redox Engineering of Myoglobin by Cofactor Substitution to Enhance Cyclopropanation Reactivity.

Design of metal cofactor ligands is essential for controlling the reactivity of metalloenzymes. We investigated a carbene transfer reaction catalyzed by myoglobins containing iron porphyrin cofactors with one and two trifluoromethyl groups at peripheral sites (FePorCF3 and FePor(CF3)2, respectively), native heme and iron porphycene (FePc). These four myoglobins show a wide range of Fe(II)/Fe(III) redox potentials in the protein of +147 mV, +87 mV, +42 mV and -198 mV vs. NHE, respectively. Myoglobin reconstituted with FePor(CF3)2 has a more positive potential, which enhances the reactivity of a carbene intermediate with alkenes, and demonstrates superior cyclopropanation of inert alkenes, such as aliphatic and internal alkenes. In contrast, engineered myoglobin reconstituted with FePc has a more negative redox potential, which accelerates the formation of the intermediate, but has low reactivity for inert alkenes. Mechanistic studies indicate that myoglobin with FePor(CF3)2 generates an undetectable active intermediate with a radical character. In contrast, this reaction catalyzed by myoglobin with FePc includes a detectable iron-carbene species with electrophilic character. This finding highlights the importance of redox-focused design of the iron porphyrinoid cofactor in hemoproteins to tune the reactivity of the carbene transfer reaction.

Focusing on a nickel hydrocorphinoid in a protein matrix: methane generation by methyl-coenzyme M reductase with F430 cofactor and its models.

Methyl-coenzyme M reductase (MCR) containing a nickel hydrocorphinoid cofactor, F430, is an essential enzyme that catalyzes anaerobic methane generation and oxidation. The active Ni(I) species in MCR converts methyl-coenzyme M (CH3S-CoM) and coenzyme B (HS-CoB) to methane and heterodisulfide (CoM-S-S-CoB). Extensive experimental and theoretical studies focusing on the substrate-binding cavity including the F430 cofactor in MCR have suggested two principally different reaction mechanisms involving an organonickel CH3-Ni(III) species or a transient methyl radical species. In parallel with research on native MCR itself, the functionality of MCR has been investigated in the context of model complexes of F430 and recent protein-based functional models, which include a nickel complex. In the latter case, hemoproteins reconstituted with tetradehydro- and didehydrocorrinoid nickel complexes have been found to represent useful model systems that are responsible for methane generation. These efforts support the proposed mechanism of the enzymatic reaction and provide important insight into replicating the MCR-like methane-generation process. Furthermore, the modeling of MCR described here is expected to lead to understanding of protein-supported nickel porphyrinoid chemistry as well as the creation of MCR-inspired catalysis.

Reactivity of Myoglobin Reconstituted with Cobalt Corrole toward Hydrogen Peroxide.

The protein matrix of natural metalloenzymes regulates the reactivity of metal complexes to establish unique catalysts. We describe the incorporation of a cobalt complex of corrole (CoCor), a trianionic porphyrinoid metal ligand, into an apo-form of myoglobin to provide a reconstituted protein (rMb(CoCor)). This protein was characterized by UV-vis, EPR, and mass spectroscopic measurements. The reaction of rMb(CoCor) with hydrogen peroxide promotes an irreversible oxidation of the CoCor cofactor, whereas the same reaction in the presence of a phenol derivative yields the cation radical form of CoCor. Detailed kinetic investigations indicate the formation of a transient hydroperoxo complex of rMb(CoCor) which promotes the oxidation of the phenol derivatives. This mechanism is significantly different for native heme-dependent peroxidases, which generate a metal-oxo species as an active intermediate in a reaction with hydrogen peroxide. The present findings of unique reactivity will contribute to further design of artificial metalloenzymes.

DNA-Mediated Protein Shuttling between Coacervate-Based Artificial Cells.

The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single-stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA-modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures.

Functional Myoglobin Model Composed of a Strapped Porphyrin/Cyclodextrin Supramolecular Complex with an Overhanging COOH That Increases O2/CO Binding Selectivity in Aqueous Solution.

A water-soluble strapped iron(III)tetraarylporphyrin (FeIIIPor-1) bearing two propylpyridinium groups at the side chains and a carboxylic acid group at the overhanging position of the strap was synthesized to mimic the function of myoglobin with the distal polar functionality in aqueous solution. FeIIIPor-1 forms a stable 1:1 inclusion complex with a per-O-methylated ß-cyclodextrin dimer having a pyridine linker (Py3OCD), providing a hydrophobic environment and a proximal fifth ligand to stabilize the O2-complex. The ferrous complex (FeIIPorCD-1) binds both O2 and CO in aqueous solution. The O2 and CO binding affinities (P1/2O2 and P1/2CO) and half-life time (t1/2) of the O2 complex of FeIIPorCD-1 are 6.3 and 0.021 Torr, and 7 h, respectively, at pH 7 and 25 °C. The control compound without the strap structure (FeIIPorCD-2) has similar oxygen binding characteristics (P1/2O2 = 8.0 Torr), but much higher CO binding affinity (P1/2CO = 3.8 × 10-4 Torr), and longer t1/2 (30 h). The O2 and CO kinetics indicate that the strapped structure in FeIIPorCD-1 inhibits the entrance of these gaseous ligands into the iron(II) center, as evidenced by lower konO2 and konCO values. Interestingly, the CO complex of FeIIPorCD-1 is significantly destabilized (relatively larger koffCO), while the koffO2 value is much smaller than that of FeIIPorCD-2, resulting in significantly increased O2/CO selectivity (reduced M value, where M = P1/2O2/P1/2CO = 320) in FeIIPorCD-1 compared to FeIIPorCD-2 (M = 21000).

A Supramolecular Assembly of Hemoproteins Formed in a Star-Shaped Structure via Heme-Heme Pocket Interactions.

Proteins have been used as building blocks to provide various supramolecular structures in efforts to develop nano-biomaterials possessing broad biological functionalities. A series of unique structures have been obtained from the engineering of hemoproteins which contain the iron porphyrin known as heme, as a prosthetic group. This work in developing assembling systems is extended using cytochrome b562, a small electron transfer hemoprotein engineered to include an externally-attached heme moiety. The engineered units, which form a one-dimensional assembly via interprotein heme-heme pocket interactions, are conjugated to an apo-form of hexameric tyrosine-coordinated hemoprotein (apoHTHP) to provide a branching unit promoting the assembly of a star-shaped structure. The incorporation of the heme moiety attached to the protein surface of cytochrome b562 into apoHTHP can be accelerated by elevating the reaction temperature to generate a new assembly. The formation of a new larger assembly structure was confirmed by size exclusion chromatography. The ratio of the heme-containing units in the assemblies was analyzed by UV-Vis spectroscopy and the population of protein units estimated from SDS PAGE suggests the presence of plausible star-shaped structures, which are supported by hydrodynamic diameter data obtained by dynamic light scattering.

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA-Based Recruitment Domains.

Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase-9, using short DNA elements as modular recruitment domains. Caspase-9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase-9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly(N-isopropylacrylamide) toward an Artificial Light-Harvesting System.

Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly(N-isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

Hemoproteins Reconstituted with Artificial Metal Complexes as Biohybrid Catalysts.

In nature, heme cofactor-containing proteins participate not only in electron transfer and O2 storage and transport but also in biosynthesis and degradation. The simplest and representative cofactor, heme b, is bound within the heme pocket via noncovalent interaction in many hemoproteins, suggesting that the cofactor is removable from the protein, leaving a unique cavity. Since the cavity functions as a coordination sphere for heme, it is of particular interest to investigate replacement of native heme with an artificial metal complex, because the substituted metal complex will be stabilized in the heme pocket while providing alternative chemical properties. Thus, cofactor substitution has great potential for engineering of hemoproteins with alternative functions. For these studies, myoglobin has been a focus of our investigations, because it is a well-known oxygen storage hemoprotein. However, the heme pocket of myoglobin has been only arranged for stabilizing the heme-bound dioxygen, so the structure is not suitable for activation of small molecules such as H2O2 and O2 as well as for binding an external substrate. Thus, the conversion of myoglobin to an enzyme-like biocatalyst has presented significant challenges. The results of our investigations have provided useful information for chemists and biologists. Our own efforts to develop functionalized myoglobin have focused on the incorporation of a chemically modified cofactor into apomyoglobin in order to (1) construct an artificial substrate-binding site near the heme pocket, (2) increase cofactor reactivity, or (3) promote a new reaction that has never before been catalyzed by a native heme enzyme. In pursuing these objectives, we first found that myoglobin reconstituted with heme having a chemically modified heme-propionate side chain at the exit of the heme pocket has peroxidase activity with respect to oxidation of phenol derivatives. Our recent investigations have succeeded in enhancing oxidation and oxygenation activities of myoglobin as well as promoting new reactions by reconstitution of myoglobin with new porphyrinoid metal complexes. Incorporation of suitable metal porphyrinoids into the heme pocket has produced artificial enzymes capable of efficiently generating reactive high valent metal-oxo and metallocarbene intermediates to achieve the catalytic hydroxylation of C(sp3)-H bonds and cyclopropanation of olefin molecules, respectively. In other efforts, we have focused on nitrobindin, an NO-binding hemoprotein, because aponitrobindin includes a ß-barrel cavity, which provides a robust structure highly similar to that of the native holoprotein. It was expected that the aponitrobindin would be suitable for development as a protein scaffold for a metal complex. Recently, it was confirmed that several organometallic complexes can bind to this scaffold and function as catalysts promoting hydrogen evolution or C-C bond formation. The hydrophobic ß-barrel structure plays a significant role in substrate binding as well as controlling the stereoselectivity of the reactions. Furthermore, these catalytic activities and stereoselectivities are remarkably improved by mutation-dependent modifications of the cavity structure for the artificial cofactor. This Account demonstrates how apoproteins of hemoproteins can provide useful protein scaffolds for metal complexes. Further development of these concepts will provide a useful strategy for generation of robust and useful artificial metalloenzymes.

Methane Generation and Reductive Debromination of Benzylic Position by Reconstituted Myoglobin Containing Nickel Tetradehydrocorrin as a Model of Methyl-coenzyme M Reductase.

Methyl-coenzyme M reductase (MCR), which contains the nickel hydrocorphinoid cofactor F430, is responsible for biological methane generation under anaerobic conditions via a reaction mechanism which has not been completely elucidated. In this work, myoglobin reconstituted with an artificial cofactor, nickel(I) tetradehydrocorrin (NiI(TDHC)), is used as a protein-based functional model for MCR. The reconstituted protein, rMb(NiI(TDHC)), is found to react with methyl donors such as methyl p-toluenesulfonate and trimethylsulfonium iodide with methane evolution observed in aqueous media containing dithionite. Moreover, rMb(NiI(TDHC)) is found to convert benzyl bromide derivatives to reductively debrominated products without homocoupling products. The reactivity increases in the order of primary > secondary > tertiary benzylic carbons, indicating steric effects on the reaction of the nickel center with the benzylic carbon in the initial step. In addition, Hammett plots using a series of para-substituted benzyl bromides exhibit enhancement of the reactivity with introduction of electron-withdrawing substituents, as shown by the positive slope against polar substituent constants. These results suggest a nucleophilic SN2-type reaction of the Ni(I) species with the benzylic carbon to provide an organonickel species as an intermediate. The reaction in D2O buffer at pD 7.0 causes a complete isotope shift of the product by +1 mass unit, supporting our proposal that protonation of the organonickel intermediate occurs during product formation. Although the turnover numbers are limited due to inactivation of the cofactor by side reactions, the present findings will contribute to elucidating the reaction mechanism of MCR-catalyzed methane generation from activated methyl sources and dehalogenation.

Myoglobin Reconstituted with Ni Tetradehydrocorrin as a Methane-Generating Model of Methyl-coenzyme M Reductase.

Myoglobin reconstituted with Ni tetradehydrocorrin was investigated as a model of F430-containing methyl-coenzyme M reductase, which catalyzes anaerobic methane generation. The NiII tetradehydrocorrin complex has a NiII /NiI redox potential of -0.34 V vs. SHE and EPR spectroscopy indicates the formation of a NiI species upon reduction by dithionite. This redox potential is approximately 0.31 V more positive than that of F430. The NiI tetradehydrocorrin moiety is bound to the apo-form of myoglobin to yield the reconstituted protein. Methane gas is generated in the reaction of the model with methyl iodide in the presence of the reconstituted protein under reductive conditions, whereas the NiI complex itself does not produce methane gas. This is the first example of a protein-based functional model of F430-containing methyl-coenzyme M reductase.

Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme-Heme Exciton Coupling.

A supramolecular assembly of units of cytochrome b562 with externally attached heme having intermolecular linkages formed via the heme-heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome b562 provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme-heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme-heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly.

Synthesis and Characterization of meso-Substituted Cobalt Tetradehydrocorrin and Evaluation of Its Electrocatalytic Behavior Toward CO2 Reduction and H2 Evolution.

A meso-aryl substituted cobalt(II) tetradehydrocorrin complex (Co(II)TDHC) has been synthesized and investigated. The corrin framework, determined by X-ray crystallographic analysis, is found to be relatively planar except at the C1 and C19 positions. Cyclic voltammetry (CV) measurements indicate two positively shifted reversible redox couples at -0.53 and -1.70 V vs Fc/Fc+ for [CoII]+/[CoI] and [CoI]/([CoI]•- and/or [CoII]-) ([CoII] = Co(II)TDHC), respectively, compared with the previously reported cobalt porphyrin complex, because the tetradehydrocorrin ligand efficiently promotes the formation of low-valent metal species due to its monoanionic character. Furthermore, it is found that the current in the CV measurement is significantly enhanced upon addition of H2O under a CO2 atmosphere, indicating the progression of electroreductive catalysis by Co(II)TDHC. However, controlled-potential electrolysis (CPE) using Co(II)TDHC under the same conditions shows generation of H2 as a major product and only a small amount of CO as a CO2 reduction product; Faradaic efficiencies are calculated to be 66.8 and 4.5%, respectively. The CPE with a buffer solution under an N2 atmosphere reveals that the selective H2 generation is promoted by the moderate acidification of the solution under CO2 saturation conditions. The present study demonstrates that the significantly stabilized Co(I) species with the monoanionic ligand framework preferentially catalyzes the thermodynamically favored H2 evolution rather than CO2 reduction.

Successive energy transfer within multiple photosensitizers assembled in a hexameric hemoprotein scaffold.

An assembly of multiple photosensitizers is demonstrated by development of a hexameric hemoprotein (HTHP) scaffold as a light harvesting model to replicate the successive energy transfer occuring within photosensitizer assemblies of natural systems. In our model, six zinc protoporphyrin IX (ZnPP) molecules are arrayed at the heme binding site of HTHP by supramolecular interactions and five fluorescein (Flu) molecules and one Texas Red (Tex) molecule as donor and acceptor photosensitizers, respectively, are attached to the HTHP protein surface with covalent linkages. The flow of excited energy from photoexcited Flu to Tex occurs via two pathways: direct energy transfer from Flu to Tex (path 1) and energy transfer via ZnPP (path 2). Steady state and time-resolved fluorescence measurements reveal that the energy transfer ratio of these pathways (path 1 : path 2) is 39 : 61. These findings indicate that the excited energy originating at five Flu and six ZnPP molecules is collected at one Tex molecule as a funnel-like bottom for light harvesting. The present system using the hexameric hemoprotein scaffold is a promising candidate for construction of an artificial light harvesting system having multiple photosensitizers to promote efficient use of solar energy.

Manganese(V) Porphycene Complex Responsible for Inert C-H Bond Hydroxylation in a Myoglobin Matrix.

A mechanistic study of H2O2-dependent C-H bond hydroxylation by myoglobin reconstituted with a manganese porphycene was carried out. The X-ray crystal structure of the reconstituted protein obtained at 1.5 Å resolution reveals tight incorporation of the complex into the myoglobin matrix at pH 8.5, the optimized pH value for the highest turnover number of hydroxylation of ethylbenzene. The protein generates a spectroscopically detectable two-electron oxidative intermediate in a reaction with peracid, which has a half-life up to 38 s at 10 °C. Electron paramagnetic resonance spectra of the intermediate with perpendicular and parallel modes are silent, indicating formation of a low-spin MnV-oxo species. In addition, the MnV-oxo species is capable of promoting the hydroxylation of sodium 4-ethylbenzenesulfonate under single turnover conditions with an apparent second-order rate constant of 2.0 M-1 s-1 at 25 °C. Furthermore, the higher bond dissociation enthalpy of the substrate decreases the rate constant, in support of the proposal that the H-abstraction is one of the rate-limiting steps. The present engineered myoglobin serves as an artificial metalloenzyme for inert C-H bond activation via a high-valent metal species similar to the species employed by native monooxygenases such as cytochrome P450.

Catalytic Cyclopropanation by Myoglobin Reconstituted with Iron Porphycene: Acceleration of Catalysis due to Rapid Formation of the Carbene Species.

Myoglobin reconstituted with iron porphycene catalyzes the cyclopropanation of styrene with ethyl diazoacetate. Compared to native myoglobin, the reconstituted protein significantly accelerates the catalytic reaction and the kcat/Km value is 26-fold enhanced. Mechanistic studies indicate that the reaction of the reconstituted protein with ethyl diazoacetate is 615-fold faster than that of native myoglobin. The metallocarbene species reacts with styrene with the apparent second-order kinetic constant of 28 mM-1 s-1 at 25 °C. Complementary theoretical studies support efficient carbene formation by the reconstituted protein that results from the strong ligand field of the porphycene and fewer intersystem crossing steps relative to the native protein. From these findings, the substitution of the cofactor with an appropriate metal complex serves as an effective way to generate a new biocatalyst.

Cobalt tetradehydrocorrins coordinated by imidazolate-like histidine in the heme pocket of horseradish peroxidase.

Horseradish peroxidase was reconstituted with cobalt tetradehydrocorrin, rHRP(Co(TDHC)), as a structural analog of cobalamin coordinated with an imidazolate-like His residue, which is generally seen in native enzymes. In contrast to the previously reported cobalt tetradehydrocorrin-reconstituted myoglobin, rMb(Co(TDHC)), the HRP matrix was expected to provide strong axial ligation by His170 which has imidazolate character. rHRP(CoII(TDHC)) was characterized by EPR and its reaction with reductants indicates a negative shift of its redox potential compared to rMb(Co(TDHC)). Furthermore, aqua- and CN-forms of Co(III) state were prepared. The former species was obtained by oxidation of rHRP(CoII(TDHC)) with K3[Fe(CN)6]. The cyanide-coordinated Co(III) species in the latter was prepared by ligand exchange of rHRP(CoIII(OH)(TDHC)) with exogenous cyanide upon addition of KCN. The 13C NMR chemical shift of cyanide in rHRP(CoIII(CN)(TDHC)) was determined to be 121.8 ppm. IR measurements show that the cyanide of rHRP(CoIII(CN)(TDHC)) has a stretching frequency peak at 2144 cm-1. The 13C NMR and IR measurements indicate strong coordination of cyanide to CoIII(TDHC) relative to rMb(CoIII(CN)(TDHC)). Thus, the extent of π-back donation from the cobalt ion to the cyanide ion is relatively high in rHRP(CoIII(CN)(TDHC)). The pK 1/2 values of rHRP(CoIII(OH)(TDHC)) and rHRP(CoIII(CN)(TDHC)) are the same (pK 1/2 = 3.2) as determined by a pH titration experiment, indicating that cyanide ligation does not affect Co-His ligation, whereas cyanide ligation weakens the Co-His ligation in rMb(CoIII(CN)(TDHC)). Taken together, these results indicate that HRP reconstituted with cobalt tetradehydrocorrin is a suitable cobalamin-dependent enzyme model with imidazolate-like His residue.

CuAAC in a Distal Pocket: Metal Active-Template Synthesis of Strapped-Porphyrin [2]Rotaxanes.

The synthesis of a porphyrin rotaxane by dipolar cycloaddition takes advantage of the ditopic character of a phenanthroline-strapped porphyrin. The success of the click reaction was conditioned by the presence of both a coordinatively unsaturated metal in the porphyrin and a copper(I) bound to the phenanthroline, pointing at a new "tandem active metal template" mechanism.

Iron-Strapped Porphyrins with Carboxylic Acid Groups Hanging over the Coordination Site: Synthesis, X-ray Characterization, and Dioxygen Binding.

A series of myoglobin active site analogues were synthesized and characterized to investigate the dioxygen binding effects of a flexible distal strap over the coordination site. These four synthetic models differ mostly by the shape and polarity of their cavities and also possibly by motion of the distal strap attached to two of the meso carbon atoms. Each of the four models has an intramolecular nitrogen base that axially binds the iron(II) cation inside the porphyrin, but they differ either by the nature of the distal strap or by its mobility. The overhanging distal group is either a generally apolar ethyl malonate group or a polar malonic acid group which is also a strong H-bond donor. It is shown that, in the ferrous complex 2b bearing such an overhung malonic acid group in close proximity to the iron atom, the equilibrium rate for dioxygen binding is significantly enhanced in comparison to that of its ester precursor. In the case of the analogous complex 1b bearing a more mobile distal strap, one of the carboxylic acid groups binds the iron(II) cation, leading to a six-coordinate ferrous complex. Unexpectedly, this complex proved to be high-spin (S = 2) as shown by solid-state magnetic measurements. Whereas this unprecedented complex still binds dioxygen, the formation of the intramolecular six-coordinate complex precluded the measurement of its dioxygen affinity through direct quantitative gas titration monitored by UV-vis spectroscopy. However, in this case, the determination of the kinetic rate constants for dioxygen binding and dissociation by laser flash photolysis allowed the evaluation of the equilibrium rate. Together with three previous X-ray structures of iron complexes in the ααßß conformation, the structure of the cavity and the shape of the relaxed distal strap are also discussed with the consideration of the resolution of X-ray structures of two different free-base ligands in the ααßß conformation, with one bearing the ethyl malonate group and the second one bearing the malonic acid group. A third X-ray structure of the analogous ligand with the overhanging ethyl malonate group in the αßαß series allows a direct comparison of the distal strap in both geometries. This work reveals that the compound with the overhanging carboxylic acid group which cannot directly interact with the ferrous heme exhibits an increased dioxygen affinity by 2 orders of magnitude versus its ester precursor.

Redox Potentials of Cobalt Corrinoids with Axial Ligands Correlate with Heterolytic Co-C Bond Dissociation Energies.

We investigate the correlations between the redox potentials of nonalkylated cobalt corrinoids and the Co-C bond dissociation energies (BDEs) of the methylated species with an aqua or histidine axial ligand. A set of cobalt corrinoids, cobalamin, and its model systems, which include new version of myoglobin reconstituted with cobalt didehydrocorrin, are investigated. The Co(III)/Co(II) and Co(II)/Co(I) redox potentials of myoglobin reconstituted with cobalt tetradehydrocorrin and didehydrocorrin and the bare cofactors were determined. Density functional theory (DFT) calculations were performed to estimate the Co-C BDEs of the methylated species. It is found that the redox potentials correlate well with the heterolytic BDEs, which are dependent on the electronegativity of the corrinoid frameworks. The present study offers two important insights into our understanding of how enzymes promote the reactions: (i) homolysis is promoted by strong axial ligation and (ii) heterolysis is controlled by the redox potentials, which are regulated by the saturated framework and axial ligation in the enzyme.