Troy A. Stich

EPR Evidence for Co(IV) Species Produced During Water Oxidation at Neutral pH

Thin-film water oxidation catalysts (Co-Pi) prepared by electrodeposition from phosphate electrolyte and Co(NO(3))(2) have been characterized by electron paramagnetic resonance (EPR) spectroscopy. Co-Pi catalyst films exhibit EPR signals corresponding to populations of both Co(II) and Co(IV). As the deposition voltage is increased into the region where water oxidation prevails, the population of Co(IV) rises and the population of Co(II) decreases. The changes in the redox speciation of the film can also be induced, in part, by prolonged water oxidation catalysis in the absence of additional catalyst deposition. These results provide spectroscopic evidence for the formation of Co(IV) species during water oxidation catalysis at neutral pH.

Electronic Structure Description of a [Co(III)3Co(IV)O4] Cluster: A Model for the Paramagnetic Intermediate in Cobalt-Catalyzed Water Oxidation

Multifrequency electron paramagnetic resonace (EPR) spectroscopy and electronic structure calculations were performed on [Co(4)O(4)(C(5)H(5)N)(4)(CH(3)CO(2))(4)](+) (1(+)), a cobalt tetramer with total electron spin S = 1/2 and formal cobalt oxidation states III, III, III, and IV. The cuboidal arrangement of its cobalt and oxygen atoms is similar to that of proposed structures for the molecular cobaltate clusters of the cobalt-phosphate (Co-Pi) water-oxidizing catalyst. The Davies electron-nuclear double resonance (ENDOR) spectrum is well-modeled using a single class of hyperfine-coupled (59)Co nuclei with a modestly strong interaction (principal elements of the hyperfine tensor are equal to [-20(±2), 77(±1), -5(±15)] MHz). Mims (1)H ENDOR spectra of 1(+) with selectively deuterated pyridine ligands confirm that the amount of unpaired spin on the cobalt-bonding partner is significantly reduced from unity. Multifrequency (14)N ESEEM spectra (acquired at 9.5 and 34.0 GHz) indicate that four nearly equivalent nitrogen nuclei are coupled to the electron spin. Cumulatively, our EPR spectroscopic findings indicate that the unpaired spin is delocalized almost equally across the eight core atoms, a finding corroborated by results from DFT calculations. Each octahedrally coordinated cobalt ion is forced into a low-spin electron configuration by the anionic oxo and carboxylato ligands, and a fractional electron hole is localized on each metal center in a Co 3d(xz,yz)-based molecular orbital for this essentially [Co(+3.125)(4)O(4)] system. Comparing the EPR spectrum of 1(+) with that of the catalyst film allows us to draw conclusions about the electronic structure of this water-oxidation catalyst.

A High-Spin Iron(IV)–Oxo Complex Supported by a Trigonal Nonheme Pyrrolide Platform

We report the generation and characterization of a new high-spin iron(IV)-oxo complex supported by a trigonal nonheme pyrrolide platform. Oxygen-atom transfer to [(tpa(Mes))Fe(II)](-) (tpa(Ar) = tris(5-arylpyrrol-2-ylmethyl)amine) in acetonitrile solution affords the Fe(III)-alkoxide product [(tpa(Mes2MesO))Fe(III)](-) resulting from intramolecular C-H oxidation with no observable ferryl intermediates. In contrast, treatment of the phenyl derivative [(tpa(Ph))Fe(II)](-) with trimethylamine N-oxide in acetonitrile solution produces the iron(IV)-oxo complex [(tpa(Ph))Fe(IV)(O)](-) that has been characterized by a suite of techniques, including mass spectrometry as well as UV-vis, FTIR, Mössbauer, XAS, and parallel-mode EPR spectroscopies. Mass spectral, FTIR, and optical absorption studies provide signatures for the iron-oxo chromophore, and Mössbauer and XAS measurements establish the presence of an Fe(IV) center. Moreover, the Fe(IV)-oxo species gives parallel-mode EPR features indicative of a high-spin, S = 2 system. Preliminary reactivity studies show that the high-spin ferryl tpa(Ph) complex is capable of mediating intermolecular C-H oxidation as well as oxygen-atom transfer chemistry.

The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster.

Three iron-sulfur proteins–HydE, HydF, and HydG–play a key role in the synthesis of the [2Fe]H component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-l-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN− ligands of the [2Fe]H cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN− ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the 13C, 15N, and 57Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe]H component of the H-cluster. Vibrational spectroscopy traces the origin of carbon monoxide and cyanide ligands in the active site of di-iron hydrogenase enzymes. [Also see Perspective by Pickett] Sourcing CO and Cyanide Hydrogenase enzymes derive their activity in part from the coordination of CO and cyanide ligands to metals in their active sites. Recent work elucidated the jettisoning of a tyrosine side chain at the outset of the biosynthetic pathway toward these ligands in the di-iron class of hydrogenase. Kuchenreuther et al. (p. 424; see the Perspective by Pickett) now apply stopped-flow infrared spectroscopy to uncover the next portion of the pathway, during which the residual tyrosine fragment is further broken down into CO and CN− ligands at a single iron center in an iron sulfur cluster associated with the HydG enzyme.

A Radical Intermediate in Tyrosine Scission to the CO and CN− Ligands of FeFe Hydrogenase

Piecing Together Hydrogenase Microbial hydrogenase enzymes generally use iron to catalyze the reversible formation of hydrogen from protons and electrons. Key to their efficiency is a set of iron-coordinating ligands, including CO and cyanide. Kuchenreuther et al. (p. 472) examined how the HydG maturase enzyme breaks down the amino acid tyrosine to derive these diatomic ligands for assembly of the diiron class of hydrogenases. The first step involves abstraction of an H atom from the phenolic OH substituent of the side chain. Electron paramagnetic resonance spectroscopy revealed a radical intermediate that subsequently results from heterolysis of the bond tethering the side chain to the α-carbon. With the side chain thus jettisoned, the residual dehydroglycine could be transformed into CO and CN−. Electron paramagnetic resonance spectroscopy elucidates a key step in the biosynthesis of hydrogenase active site ligands. The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN− for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of 2H, 13C, and 15N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cβ bond forms a transient 4-oxidobenzyl (4OB•) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB• radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN−, a key intermediate in the assembly of the 2Fe subunit of the H cluster.

Direct Spectroscopic Observation of Large Quenching of First Order Orbital Angular Momentum with Bending in Monomeric, Two-Coordinate Fe(II) Primary Amido Complexes and the Profound Magnetic Effects of the Absence of Jahn- and Renner-Teller Distortions in Rigorously Linear Coordination

The monomeric iron(II) amido derivatives Fe{N(H)Ar*}(2) (1), Ar* = C(6)H(3)-2,6-(C(6)H(2)-2,4,6-Pr(i)(3))(2), and Fe{N(H)Ar(#)}(2) (2), Ar(#) = C(6)H(3)-2,6-(C(6)H(2)-2,4,6-Me(3))(2), were synthesized and studied in order to determine the effects of geometric changes on their unusual magnetic properties. The compounds, which are the first stable homoleptic primary amides of iron(II), were obtained by the transamination of Fe{N(SiMe(3))(2)}(2), with HN(SiMe(3))(2) elimination, by the primary amines H(2)NAr* or H(2)NAr(#). X-ray crystallography showed that they have either strictly linear (1) or bent (2, N-Fe-N = 140.9(2) degrees ) iron coordination. Variable temperature magnetization and applied magnetic field Mossbauer spectroscopy studies revealed a very large dependence of the magnetic properties on the metal coordination geometry. At ambient temperature, the linear 1 displayed an effective magnetic moment in the range 7.0-7.50 mu(B), consistent with essentially free ion magnetism. There is a very high internal orbital field component, H(L) approximately 170 T which is only exceeded by a H(L) approximately 203 T of Fe{C(SiMe(3))(3)}(2). In contrast, the strongly bent 2 displayed a significantly lower mu(eff) value in the range 5.25-5.80 mu(B) at ambient temperature and a much lower orbital field H(L) value of 116 T. The data for the two amido complexes demonstrate a very large quenching of the orbital magnetic moment upon bending the linear geometry. In addition, a strong correlation of H(L) with overall formal symmetry is confirmed. ESR spectroscopy supports the existence of large orbital magnetic moments in 1 and 2, and DFT calculations provide good agreement with the physical data.

Biochemical and EPR-Spectroscopic Investigation into Heterologously Expressed Vinyl Chloride Reductive Dehalogenase (VcrA) from Dehalococcoides mccartyi Strain VS

Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.

Ligation of D1-His332 and D1-Asp170 to the Manganese Cluster of Photosystem II from Synechocystis Assessed by Multifrequency Pulse EPR Spectroscopy

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is used to ascertain the nature of the bonding interactions of various active site amino acids with the Mn ions that compose the oxygen-evolving cluster (OEC) in photosystem II (PSII) from the cyanobacterium Synechocystis sp. PCC 6803 poised in the S(2) state. Spectra of natural isotopic abundance PSII ((14)N-PSII), uniformly (15)N-labeled PSII ((15)N-PSII), and (15)N-PSII containing (14)N-histidine ((14)N-His/(15)N-PSII) are compared. These complementary data sets allow for a precise determination of the spin Hamiltonian parameters of the postulated histidine nitrogen interaction with the Mn ions of the OEC. These results are compared to those from a similar study on PSII isolated from spinach. Upon mutation of His332 of the D1 polypeptide to a glutamate residue, all isotopically sensitive spectral features vanish. Additional K(a)- and Q-band ESEEM experiments on the D1-D170H site-directed mutant give no indication of new (14)N-based interactions.

Ammonia Binds to the Dangler Manganese of the Photosystem II Oxygen-Evolving Complex

High-resolution X-ray structures of photosystem II reveal several potential substrate binding sites at the water-oxidizing/oxygen-evolving 4MnCa cluster. Aspartate-61 of the D1 protein hydrogen bonds with one such water (W1), which is bound to the dangler Mn4A of the oxygen-evolving complex. Comparison of pulse EPR spectra of (14)NH3 and (15)NH3 bound to wild-type Synechocystis PSII and a D1-D61A mutant lacking this hydrogen-bonding interaction demonstrates that ammonia binds as a terminal NH3 at this dangler Mn4A site and not as a partially deprotonated bridge between two metal centers. The implications of this finding on identifying the binding sites of the substrate and the subsequent mechanism of dioxygen formation are discussed.

Multifrequency EPR studies of manganese catalases provide a complete description of proteinaceous nitrogen coordination.

Pulse electron paramagnetic resonance (EPR) spectroscopy is employed at two very different excitation frequencies, 9.77 and 30.67 GHz, in the study of the nitrogen coordination environment of the Mn(III)Mn(IV) state of the dimanganese-containing catalases from Lactobacillus plantarum and Thermus thermophilus. Consistent with previous studies, the lower-frequency results reveal one unique histidine nitrogen-Mn cluster interaction. For the first time, a second, more strongly hyperfine-coupled (14)N atom is unambiguously observed through the use of higher frequency/higher field EPR spectroscopy. The low excitation frequency spectral features are rationalized as arising from the interaction of a histidine nitrogen that is bound to the Mn(IV) ion, and the higher excitation frequency features are attributed to the histidine nitrogen bound to the Mn(III) ion. These results allow for the computation of intrinsic hyperfine coupling constants, which range from 2.2 to 2.9 MHz, for sp(2)-hybridized nitrogens coordinating equatorially to high-valence Mn ions. The relevance of these findings is discussed in the context of recent results from analogous higher frequency EPR studies of the Mn cluster in photosystem II and other exchange-coupled, transition metal-containing systems.