Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S; 112; E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic Fe; II; complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S; 112; E. Two aqua ligands complete the primary coordination sphere of the Fe; II; center with both involved in hydrogen bond networks within Sav. The corresponding Fe; III; protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The Fe; III; protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD

Preparation of Non-heme {FeNO}⁷ Models of Cysteine Dioxygenase: Sulfur versus Nitrogen Ligation and Photorelease of Nitric Oxide

We present the synthesis and spectroscopic characterization of [Fe­(NO)­(N3PyS)]­BF₄ (<b>3</b>), the first structural and electronic model of NO-bound cysteine dioxygenase. The nearly isostructural all-N-donor analogue [Fe­(NO)­(N4Py)]­(BF₄)₂ (<b>4</b>) was also prepared, and comparisons of <b>3</b> and <b>4</b> provide insight regarding the influence of S vs N ligation in {FeNO}⁷ species. One key difference occurs upon photoirradiation, which causes the fully reversible release of NO from <b>3</b>, but not from <b>4</b>

Preparation of Non-heme {FeNO}⁷ Models of Cysteine Dioxygenase: Sulfur versus Nitrogen Ligation and Photorelease of Nitric Oxide

We present the synthesis and spectroscopic characterization of [Fe­(NO)­(N3PyS)]­BF₄ (<b>3</b>), the first structural and electronic model of NO-bound cysteine dioxygenase. The nearly isostructural all-N-donor analogue [Fe­(NO)­(N4Py)]­(BF₄)₂ (<b>4</b>) was also prepared, and comparisons of <b>3</b> and <b>4</b> provide insight regarding the influence of S vs N ligation in {FeNO}⁷ species. One key difference occurs upon photoirradiation, which causes the fully reversible release of NO from <b>3</b>, but not from <b>4</b>

Preparation of Non-heme {FeNO}⁷ Models of Cysteine Dioxygenase: Sulfur versus Nitrogen Ligation and Photorelease of Nitric Oxide

We present the synthesis and spectroscopic characterization of [Fe­(NO)­(N3PyS)]­BF₄ (<b>3</b>), the first structural and electronic model of NO-bound cysteine dioxygenase. The nearly isostructural all-N-donor analogue [Fe­(NO)­(N4Py)]­(BF₄)₂ (<b>4</b>) was also prepared, and comparisons of <b>3</b> and <b>4</b> provide insight regarding the influence of S vs N ligation in {FeNO}⁷ species. One key difference occurs upon photoirradiation, which causes the fully reversible release of NO from <b>3</b>, but not from <b>4</b>

Preparation of Non-heme {FeNO}⁷ Models of Cysteine Dioxygenase: Sulfur versus Nitrogen Ligation and Photorelease of Nitric Oxide

We present the synthesis and spectroscopic characterization of [Fe­(NO)­(N3PyS)]­BF₄ (<b>3</b>), the first structural and electronic model of NO-bound cysteine dioxygenase. The nearly isostructural all-N-donor analogue [Fe­(NO)­(N4Py)]­(BF₄)₂ (<b>4</b>) was also prepared, and comparisons of <b>3</b> and <b>4</b> provide insight regarding the influence of S vs N ligation in {FeNO}⁷ species. One key difference occurs upon photoirradiation, which causes the fully reversible release of NO from <b>3</b>, but not from <b>4</b>

Reactivity of a Cobalt(III)-Hydroperoxo Complex in Electrophilic Reactions

The reactivity of mononuclear metal-hydroperoxo adducts has fascinated researchers in many areas due to their diverse biological and catalytic processes. In this study, a mononuclear cobalt(III)-peroxo complex bearing a tetradentate macrocyclic ligand, [Co-III(Me-3-TPADP)(O-2)](+) (Me-3-TPADP = 3,6,9- trimethy1-3,6,9-triaza-1(2,6)-pyridinacydodecaphane), was prepared by reacting [Co-II(Me-3-TPADP)(CH3CN)(2)](2+) with H2O2 in the presence of triethylamine. Upon protonation, the cobalt(III)-peroxo intermediate was converted into a cobalt (III)-hydroperoxo complex, [Co-III(Me-3-TPADP) (O2H) (CH3CN)](2+). The mononuclear cobalt(III)-peroxo and -hydroperoxo intermediates were characterized by a variety of physicochemical methods. Results of electrospray ionization mass spectrometry clearly show the transformation of the intermediates: the peak at m/z 339.2 assignable to the cobalt(III)-peroxo species disappears with concomitant growth of the peak at m/z 190.7 corresponding to the cobalt(III)-hydroperoxo complex (with bound CH3CN). Isotope labeling experiments further support the existence of the cobalt(III)-peroxo and hydroperoxo complexes. In particular, the O-O bond stretching frequency of the cobalt(III)-hydroperoxo complex was determined to be 851 cm(-1) for (O2H)-O-16 samples (803 cm(-1) for (O2H)-O-18 samples), and its Co-O vibrational energy was observed at 571 cm(-1) for (O2H)-O-16 samples (551 cm(-1) for (O2H)-O-18 samples; 568 cm(-1) for (O2H)-O-16-H-2 samples) by resonance Raman spectroscopy. Reactivity studies performed with the cobalt(III)-peroxo and hydroperoxo complexes in organic functionalizations reveal that the latter is capable of conducting oxygen atom transfer with an electrophilic character, whereas the former exhibits no oxygen atom transfer reactivity under the same reaction conditions. Alternatively, the cobalt(III)-hydroperoxo complex does not perform hydrogen atom transfer reactions, while analogous low-spin Fe(III)- hydroperoxo complexes are capable of this reactivity. Density functional theory calculations indicate that this lack of reactivity is due to the high free energy cost of O-O bond homolysis that would be required to produce the hypothetical Co(IV)-oxo product

Nuclear Resonance Vibrational Spectroscopic Definition of Peroxy Intermediates in Nonheme Iron Sites

Fe^III-(hydro)­peroxy intermediates have been isolated in two classes of mononuclear nonheme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well-defined, due to a lack of vibrational structural data. Nuclear resonance vibrational spectroscopy (NRVS) is an important technique for obtaining vibrational information on these and other intermediates, as it is sensitive to all normal modes with Fe displacement. Here, we present the NRVS spectra of side-on Fe^III-peroxy and end-on Fe^III-hydroperoxy model complexes and assign these spectra using calibrated DFT calculations. We then use DFT calculations to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe–O–O angle. This study identifies four spectroscopic handles that will enable definition of the binding mode and protonation state of Fe^III-peroxy intermediates in mononuclear nonheme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity

A Mononuclear Nonheme Iron(V)-Imido Complex

Mononuclear nonheme iron­(V)-oxo complexes have been reported previously. Herein, we report the first example of a mononuclear nonheme iron­(V)-imido complex bearing a tetraamido macrocyclic ligand (TAML), [(TAML)­Fe^V(NTs)]⁻ (<b>1</b>). The spectroscopic characterization of <b>1</b> revealed an <i>S</i> = 1/2 Fe­(V) oxidation state, an FeN bond length of 1.65(4) Å, and an FeN vibration at 817 cm^–1. The reactivity of <b>1</b> was demonstrated in CH bond functionalization and nitrene transfer reactions