MOF crystal growth: UV resonance Raman investigation of metal-ligand binding in solution and accelerated crystal growth methods.

Determining the mechanism of metal-organic framework (MOF) crystal growth is important for the development of more efficient and reliable synthetic methods. Resonance Raman spectroscopy has been used for the first time to detect interactions in solution between metal ions and bridging ligands as MOFs form. UV excitation (229 nm) produced strong resonance enhancement of 4,4'-bipyridine (bpy) vibrational bands and showed that soluble Co(2+)-bpy species formed in solution prior to the growth of MOF crystals from bpy and Co(NO3)2. The results of the Raman experiments informed the development of faster methods for synthesizing [Co2(bpy)3(NO3)4]n 2D bilayer and [Co(bpy)(NO3)2(H2O)2]n 1D chain MOFs.

Reversible heme-dependent regulation of human cystathionine ß-synthase by a flavoprotein oxidoreductase.

Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H(2)S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H(2)S, via heme-mediated allosteric regulation of CBS.

Heme regulation of human cystathionine beta-synthase activity: insights from fluorescence and Raman spectroscopy.

Cystathionine beta-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5'-phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C horizontal lineN bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O3' atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.

Modulation of the heme electronic structure and cystathionine beta-synthase activity by second coordination sphere ligands: The role of heme ligand switching in redox regulation.

In humans, cystathionine beta-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is approximately 20A away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52, respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately PLP-responsive while the R266M mutation shows dramatic differences in the ferrous state. The electrostatic interaction between C52 and R266 is critical for stabilizing the ferrous heme and its disruption leads to the facile formation of a 424nm (C-424) absorbing ferrous species, which is inactive, compared to the active 449nm ferrous species for wild-type CBS. Resonance Raman studies on the R266M mutant reveal that the kinetics of C52 rebinding after Fe-CO photolysis are comparable to that of wild-type CBS. EXAFS studies on C-424 CBS are consistent with the presence of two axial N/O low Z scatters with only one being a rigid unit of a histidine residue while the other could be a solvent molecule, an oxygen atom from the peptide backbone or a side chain nitrogen. The redox potential for the heme in full-length CBS is -350+/-4mV and is substantially lower than the value of -287+/-2mV determined for truncated CBS. A redox-regulated ligand change has the potential to serve as an allosteric on/off switch in human CBS and the second sphere ligand, R266, plays an important role in this transition.

Protein dynamics from time resolved UV Raman spectroscopy.

Raman spectroscopy can provide unique information on the evolution of structure in proteins over a wide range of time scales; the picosecond to millisecond range can be accessed with pump-probe techniques. Specific parts of the molecule are interrogated by tuning the probe laser to a resonant electronic transition, including the UV transitions of aromatic residues and of the peptide bond. Advances in laser technology have enabled the characterization of transient species at an unprecedented level of structural detail. Applications to protein unfolding and allostery are reviewed.

Investigation of an unnatural amino acid for use as a resonance Raman probe: Detection limits, solvent and temperature dependence of the νC≡N band of 4-cyanophenylalanine.

The incorporation of unnatural amino acids into proteins that act as spectroscopic probes can be used to study protein structure and function. One such probe is 4-cyanophenylalanine (PheCN), the nitrile group of which has a stretching mode that occurs in a region of the vibrational spectrum that does not contain any modes from the usual components of proteins and the wavenumber is sensitive to the polarity of its environment. In this work we evaluate the potential of UV resonance Raman spectroscopy for monitoring the sensitivity of the νC≡N band of PheCN incorporated into proteins to the protein environment. Measurement of the Raman excitation profile of PheCN showed that considerable resonance enhancement of the Raman signal was obtained using UV excitation and the best signal-to-noise ratios were obtained with excitation wavelengths of 229 and 244 nm. The detection limit for PheCN in proteins was ~10 µM, approximately a hundred-fold lower than the concentrations used in IR studies, which increases the potential applications of PheCN as a vibrational probe. The wavenumber of the PheCN νC≡N band was strongly dependent on the polarity of its environment, when the solvent was changed from H(2)O to THF it decreased by 8 cm(-1). The presence of liposomes caused a similar though smaller decrease in νC≡N for a peptide, mastoparan X, modified to contain PheCN. The selectivity and sensitivity of resonance Raman spectroscopy of PheCN mean that it can be a useful probe of intra- and intermolecular interactions in proteins and opens the door to its application in the study of protein dynamics using time-resolved resonance Raman spectroscopy.

Density functional theory analysis of molybdenum isotope fractionation.

Analytical studies have found an enrichment of the lighter Mo isotopes in oxic marine sediments compared to seawater, with isotope fractionation factors of -1.7 to -2.0 per thousand for Delta97/95Mosediment-seawater. These data place constraints on the possible identities of dissolved and adsorbed species because the equilibrium isotope fractionation depends on the energy differences between the isotopomers of the adsorbed species, minor dissolved species, and the dominant solution species, MoO42-. Adsorption likely involves molybdic acid, whose structure is indicated by previous studies to be MoO3(H2O)3. Here we used DFT calculations of vibrational frequencies to determine the isotope fractionation factors versus MoO42-. The results indicate that isotope equilibration of MoO42- with MoO3(H2O)3, yielding Delta97/95Momolybdic acid-molybdate=-1.33 per thousand, is most likely responsible for the isotope fractionation of Mo between oxic sediments and seawater. The difference between the calculated value of Delta97/95Momolybdic acid-molybdate for MoO3(H2O)3 and the value observed in natural sediments and experiments is probably due to effects of solvation and adsorption onto the manganese oxyhydroxide surface.

Dynamics of carbon monoxide binding to cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 microm(-1). Stopped flow measurements reveal slow CO association (0.0166 s(-1)) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s(-1), is essentially unchanged between pH 7.6 and 10.5, indicating that the pK(a) of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.

Reversible hydrogen gas uptake in nanoporous Prussian Blue analogues.

The family of dehydrated nanoporous Prussian Blue analogues, M(II)3[Co(III)(CN)6]2 (M(II) = Mn, Fe, Co, Ni, Cu, Zn, Cd), which contain coordinatively unsaturated divalent metal cations, undergoes reversible sorption of hydrogen gas up to 1.2 wt% (at 77 K, 101.3 kPa), the capacity of which depends on the metal ion.

Synthesis and characterization of a chromium(V) cis-dioxo bis(1,10-phenanthroline) complex and crystal and molecular structures of its chromium(III) precursor.

The first structurally characterized Cr(V) dioxo complex, cis-[CrV(O)2(phen)2](BF4) (2, phen=1,10-phenanthroline) has been synthesized by the oxidation of a related Cr(III) complex, cis-[Cr(III)(phen)2(OH2)2](NO3)3.2.5H2O (1, characterized by X-ray crystallography), with NaOCl in aqueous solutions in the presence of excess NaBF4, and its purity has been confirmed by electrospray mass spectrometry (ESMS), EPR spectroscopy, and analytical techniques. Previously reported methods for the generation of Cr(V)-phen complexes, such as the oxidation of 1 with PbO2 or PhIO, have been shown by ESMS to lead to mixtures of Cr(III), Cr(V), Cr(VI), and in some cases Cr(IV) species, 3. Species 3 was assigned as [CrIV(O)(OH)(phen)2]+, based on ESMS and X-ray absorption spectroscopy measurements. A distorted octahedral structure for 2 (CrO, 1.63 A; Cr-N, 2.04 and 2.16 A) was established by multiple-scattering (MS) modeling of XAFS spectra (solid, 10 K). The validity of the model was verified by a good agreement between the results of MS XAFS fitting and X-ray crystallography for 1 (distorted octahedron; Cr-O, 1.95 A; Cr-N, 2.06 A). Unlike for the well-studied Cr(V) 2-hydroxycarboxylato complexes, 2 was equally or more stable in aqueous media (hours at pH=1-13 and 25 degrees C) compared with polar aprotic solvents. A stable Cr(III)-Cr(VI) dimer, [Cr(III)(Cr(VI)O4)(phen)2]+ (detected by ESMS), is formed during the decomposition of 2 in nonaqueous media. Comparative studies of the oxidation of 1 by NaOCl or PbO2 have shown that [Cr(V)(O)2(phen)2]+ was the active species responsible for the previously reported oxidative DNA damage, bacterial mutagenicity, and increased incidence of micronuclei in mammalian cells, caused by the oxidation products of 1 with PbO2. Efficient oxidation of 1 to a genotoxic species, [Cr(V)(O)2(phen)2]+, in neutral aqueous media by a biological oxidant, hypochlorite, supports the hypothesis on a significant role of reoxidation of Cr(III) complexes, formed during the intracellular reduction of Cr(VI), in Cr(VI)-induced carcinogenicity. Similar oxidation reactions may contribute to the reported adverse effects of a popular nutritional supplement, Cr(III) picolinate.