Isolation of a Mixed Valence Diiron Hydride: Evidence for a Spectator Hydride in Hydrogen Evolution Catalysis

The mixed-valence diiron hydrido complex (μ-H)­Fe₂(pdt)­(CO)₂(dppv)₂ ([H<b>1</b>]⁰, where pdt =1,3-propanedithiolate and dppv = <i>cis</i>-1,2-C₂H₂(PPh₂)₂), was generated by reduction of the differous hydride [H<b>1</b>]⁺ using decamethylcobaltocene. Crystallographic analysis shows that [H<b>1</b>]⁰ retains the stereochemistry of its precursor, where one dppv ligand spans two basal sites and the other spans apical and basal positions. The Fe---Fe bond elongates to 2.80 from 2.66 Å, but the Fe–P bonds only change subtly. Although the Fe–H distances are indistinguishable in the precursor, they differ by 0.2 Å in [H<b>1</b>]⁰. The X-band electron paramagnetic resonance (EPR) spectrum reveals the presence of two stereoisomers, the one characterized crystallographically and a contribution of about 10% from a second symmetrical (<i>sym</i>) isomer wherein both dppv ligands occupy apical–basal sites. The unsymmetrical (<i>unsym</i>) arrangement of the dppv ligands is reflected in the values of <i>A</i>(³¹P), which range from 31 MHz for the basal phosphines to 284 MHz for the apical phosphine. Density functional theory calculations were employed to rationalize the electronic structure of [H<b>1</b>]⁰ and to facilitate spectral simulation and assignment of EPR parameters including ¹H and ³¹P hyperfine couplings. The EPR spectra of [H<b>1</b>]⁰ and [D<b>1</b>]⁰ demonstrate that the singly occupied molecular orbital is primarily localized on the Fe center with the longer bond to H, that is, Fe^II–H···Fe^I. The coupling to the hydride is <i>A</i>(¹H) = 55 and 74 MHz for <i>unsym</i>- amd <i>sym</i>-[H<b>1</b>]⁰, respectively. Treatment of [H<b>1</b>]⁰ with H⁺ gives 0.5 equiv of H₂ and [H<b>1</b>]⁺. Reduction of D⁺ affords D₂, leaving the hydride ligand intact. These experiments demonstrate that the bridging hydride ligand in this complex is a spectator in the hydrogen evolution reaction

Kinetics of Copper Incorporation into a Biosynthetic Purple Cu_A Azurin: Characterization of Red, Blue, and a New Intermediate Species

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple Cu_A cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of Cu_A sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the Cu_A site of a biosynthetic Cu_A model, Cu_A azurin (Cu_AAz) and found that both T1 Cu and T2 Cu intermediates form on the path to final Cu_A reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native Cu_A center of nitrous oxide reductase, the faster kinetics of copper incorporation into Cu_AAz allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ∼410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)–dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying Cu_AAz variants where the Cys and His ligands are mutated, we have identified the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of Cu_A centers

Direct EPR Observation of a Tyrosyl Radical in a Functional Oxidase Model in Myoglobin during both H₂O₂ and O₂ Reactions

Tyrosine is a conserved redox-active amino acid that plays important roles in heme–copper oxidases (HCO). Despite the widely proposed mechanism that involves a tyrosyl radical, its direct observation under O₂ reduction conditions remains elusive. Using a functional oxidase model in myoglobin called F33Y-Cu_BMb that contains an engineered tyrosine, we report herein direct observation of a tyrosyl radical during both reactions of H₂O₂ with oxidized protein and O₂ with reduced protein by electron paramagnetic resonance spectroscopy, providing a firm support for the tyrosyl radical in the HCO enzymatic mechanism

Mixed-Valence Nickel–Iron Dithiolate Models of the [NiFe]-Hydrogenase Active Site

A series of mixed-valence nickel–iron dithiolates is described. Oxidation of (diphosphine)­Ni­(dithiolate)­Fe­(CO)₃ complexes <b>1</b>, <b>2</b>, and <b>3</b> with ferrocenium salts affords the corresponding tricarbonyl cations [(dppe)­Ni­(pdt)­Fe­(CO)₃]⁺ ([<b>1</b>]⁺), [(dppe)­Ni­(edt)­Fe­(CO)₃]⁺ ([<b>2</b>]⁺) and [(dcpe)­Ni­(pdt)­Fe­(CO)₃]⁺ ([<b>3</b>]⁺), respectively, where dppe = Ph₂PCH₂CH₂PPh₂, dcpe = Cy₂PCH₂CH₂PCy₂, (Cy = cyclohexyl), pdtH₂ = HSCH₂CH₂CH₂SH, and edtH₂ = HSCH₂CH₂SH. The cation [<b>2</b>]⁺ proved unstable, but the propanedithiolates are robust. IR and EPR spectroscopic measurements indicate that these species exist as <i>C</i>_<i>s</i>-symmetric species. Crystallographic characterization of [<b>3</b>]­BF₄ shows that Ni is square planar. Interaction of [<b>1</b>]­BF₄ with P-donor ligands (L) afforded a series of substituted derivatives of type [(dppe)­Ni­(pdt)­Fe­(CO)₂L]­BF₄ for L = P­(OPh)₃ ([<b>4a</b>]­BF₄), P­(<i>p</i>-C₆H₄Cl)₃ ([<b>4b</b>]­BF₄), PPh₂(2-py) ([<b>4c</b>]­BF₄), PPh₂(OEt) ([<b>4d</b>]­BF₄), PPh₃ ([<b>4e</b>]­BF₄), PPh₂(<i>o</i>-C₆H₄OMe) ([<b>4f</b>]­BF₄), PPh₂(<i>o</i>-C₆H₄OCH₂OMe) ([<b>4g</b>]­BF₄), P­(<i>p</i>-tol)₃ ([<b>4h</b>]­BF₄), P­(<i>p</i>-C₆H₄OMe)₃ ([<b>4i</b>]­BF₄), and PMePh₂ ([<b>4j</b>]­BF₄). EPR analysis indicates that ethanedithiolate [<b>2</b>]⁺ exists as a single species at 110 K, whereas the propanedithiolate cations exist as a mixture of two conformers, which are proposed to be related through a flip of the chelate ring. Mössbauer spectra of <b>1</b> and oxidized <i>S</i> = 1/2 [<b>4e</b>]­BF₄ are both consistent with a low-spin Fe­(I) state. The hyperfine coupling tensor of [<b>4e</b>]­BF₄ has a small isotropic component and significant anisotropy. DFT calculations using the BP86, B3LYP, and PBE0 exchange–correlation functionals agree with the structural and spectroscopic data, suggesting that the SOMOs in complexes of the present type are localized in an Fe­(I)-centered d­(<i>z</i>²) orbital. The DFT calculations allow an assignment of oxidation states of the metals and rationalization of the conformers detected by EPR spectroscopy. Treatment of [<b>1</b>]⁺ with CN^– and compact basic phosphines results in complex reactions. With dppe, [<b>1</b>]⁺ undergoes quasi-disproportionation to give <b>1</b> and the diamagnetic complex [(dppe)­Ni­(pdt)­Fe­(CO)₂(dppe)]²⁺ ([<b>5</b>]²⁺), which features square-planar Ni linked to an octahedral Fe center

Mechanism of H₂ Production by Models for the [NiFe]-Hydrogenases: Role of Reduced Hydrides

The intermediacy of a reduced nickel–iron hydride in hydrogen evolution catalyzed by Ni–Fe complexes was verified experimentally and computationally. In addition to catalyzing hydrogen evolution, the highly basic and bulky (dppv)­Ni­(μ-pdt)­Fe­(CO)­(dppv) ([<b>1</b>]⁰; dppv = <i>cis</i>-C₂H₂­(PPh₂)₂) and its hydride derivatives have yielded to detailed characterization in terms of spectroscopy, bonding, and reactivity. The protonation of [<b>1</b>]⁰ initially produces <i>unsym</i>-[H<b>1</b>]⁺, which converts by a first-order pathway to <i>sym</i>-[H<b>1</b>]⁺. These species have <i>C</i>₁ (unsym) and <i>C</i>_<i>s</i> (sym) symmetries, respectively, depending on the stereochemistry of the octahedral Fe site. Both experimental and computational studies show that [H<b>1</b>]⁺ protonates at sulfur. The <i>S</i> = 1/2 hydride [H<b>1</b>]⁰ was generated by reduction of [H<b>1</b>]⁺ with Cp*₂Co. Density functional theory (DFT) calculations indicate that [H<b>1</b>]⁰ is best described as a Ni­(I)–Fe­(II) derivative with significant spin density on Ni and some delocalization on S and Fe. EPR spectroscopy reveals both kinetic and thermodynamic isomers of [H<b>1</b>]⁰. Whereas [H<b>1</b>]⁺ does not evolve H₂ upon protonation, treatment of [H<b>1</b>]⁰ with acids gives H₂. The redox state of the “remote” metal (Ni) modulates the hydridic character of the Fe­(II)–H center. As supported by DFT calculations, H₂ evolution proceeds either directly from [H<b>1</b>]⁰ and external acid or from protonation of the Fe–H bond in [H<b>1</b>]⁰ to give a labile dihydrogen complex. Stoichiometric tests indicate that protonation-induced hydrogen evolution from [H<b>1</b>]⁰ initially produces [<b>1</b>]⁺, which is reduced by [H<b>1</b>]⁰. Our results reconcile the required reductive activation of a metal hydride and the resistance of metal hydrides toward reduction. This dichotomy is resolved by reduction of the remote (non-hydride) metal of the bimetallic unit

Binuclear Cu_A Formation in Biosynthetic Models of Cu_A in Azurin Proceeds via a Novel Cu(Cys)₂His Mononuclear Copper Intermediate

Cu_A is a binuclear electron transfer (ET) center found in cytochrome <i>c</i> oxidases (C<i>c</i>Os), nitrous oxide reductases (N₂ORs), and nitric oxide reductase (NOR). In these proteins, the Cu_A centers facilitate efficient ET (<i>k</i>_ET > 10⁴ s^–1) under low thermodynamic driving forces (10–90 mV). While the structure and functional properties of Cu_A are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu_A maturation process are still lacking. Previous studies of the Cu_A assembly mechanism <i>in vitro</i> using a biosynthetic model Cu_A center in azurin (Cu_AAz) identified a novel intermediate X (I_x) during reconstitution of the binuclear site. However, because of the instability of I_x and the coexistence of other Cu centers, such as Cu_A′ and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of Cu_A assembly using variants of Glu114XCu_AAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu_A site, with a major focus on characterization of the novel intermediate I_x. We show that Cu_A assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate I_x, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → I_x formation, suggesting that Cu_A formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained I_x in ∼80–85% population in these variants, which allowed us to employ ultraviolet–visible, electron paramagnetic resonance, and extended X-ray absorption fine structure spectroscopic techniques to identify the I_x as a mononuclear Cu­(Cys)₂(His) complex. Because some of the intermediates have been proposed to be involved in the assembly of native Cu_A, these results shed light on the structural features of the important intermediates and mechanism of Cu_A formation

Redesigning the Blue Copper Azurin into a Redox-Active Mononuclear Nonheme Iron Protein: Preparation and Study of Fe(II)-M121E Azurin

Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe­(II)-M121E azurin (Az), by replacing the axial Met121 and Cu­(II) in wild-type azurin (wtAz) with Glu and Fe­(II), respectively. In contrast to the redox inactive Fe­(II)-wtAz, the Fe­(II)-M121EAz mutant can be readily oxidized by Na₂IrCl₆, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe­(II)-M121EAz, Fe­(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe­(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity

A Purple Cupredoxin from <i>Nitrosopumilus maritimus</i> Containing a Mononuclear Type 1 Copper Center with an Open Binding Site

Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon <i>Nitrosopumilus maritimus</i> SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M^–1 cm^–1) and 558 nm (2290 M^–1 cm^–1) in the UV–vis electronic spectrum. EPR studies suggest the protein contains two Cu­(II) species of nearly equal population, one nearly axial, with hyperfine constant <i>A</i>_∥ = 98 × 10^–4 cm^–1, and another more rhombic, with a smaller <i>A</i>_∥ value of 69 × 10^–4 cm^–1. The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H₂O at 2.25 Å. Both UV–vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile