Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

I. The effect of N,N,N',N' -tetramethylethylenediamine on the Schlenk Equilibrium. II. The nature of the Di-Grignard reagent

I. The influence of N,N,N’,N’-tetramethylethylenediamine on the Schlenk equilibrium

The equilibrium between ethylmagnesium bromide, diethylmagnesium, and magnesium bromide has been studied by nuclear magnetic resonance spectroscopy. The interconversion of the species is very fast on the nmr time scale, and only an averaged spectrum is observed for the ethyl species. When N,N,N’,N’-tetramethylethylenediamine is added to solutions of these reagents in tetrahydrofuran, the rate of interconversion is reduced. At temperatures near -50°, two ethylmagnesium species have been observed. These are attributed to the different ethyl groups in ethylmagnesium bromide and diethylmagnesium, two of the species involved in the Schlenk equilibrium of Grignard reagents.

II. The nature of di-Grignard reagents

Di-Grignard reagents have been examined by nuclear magnetic resonance spectroscopy in an attempt to prove that dialkylmagnesium reagents are in equilibrium with alkylmagnesium halides. The di-Grignard reagents of compounds such as 1,4-dibromobutane have been investigated. The dialkylmagnesium form of this di-Grignard reagent can exist as an intramolecular cyclic species, tetramethylene-magnesium. This cyclic form would give an nmr spectrum different from that of the classical alkylmagnesium halide di-Grignard reagent. In dimethyl ether-tetrahydrofuran solutions of di-Grignard reagents containing N N,N,N’,N’-Tetramethylethylenediamine, evidence has been found for the existence of an intramolecular dialkylmagnesium species. This species is rapidly equilibrating with other forms, but at low temperatures, the rates of interconversion are reduced. Two species can be seen in the nmr spectrum at -50°. One is the cyclic species; the other is an open form.

Inversion of the carbon at the carbon-magnesium bond in di-Grignard reagents has also been studied. This process is much faster than in corresponding monofunctional Grignard reagents.

Biological electron transfer in copper proteins

Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.

In the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.

In the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In this copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over long distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction potentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.

Radio frequency studies of surface resistance and critical magnetic field of type I and type II superconductors

The surface resistance and the critical magnetic field of lead electroplated on copper were studied at 205 MHz in a half-wave coaxial resonator. The observed surface resistance at a low field level below 4.2°K could be well described by the BCS surface resistance with the addition of a temperature independent residual resistance. The available experimental data suggest that the major fraction of the residual resistance in the present experiment was due to the presence of an oxide layer on the surface. At higher magnetic field levels the surface resistance was found to be enhanced due to surface imperfections.

The attainable rf critical magnetic field between 2.2°K and T_c of lead was found to be limited not by the thermodynamic critical field but rather by the superheating field predicted by the one-dimensional Ginzburg-Landau theory. The observed rf critical field was very close to the expected superheating field, particularly in the higher reduced temperature range, but showed somewhat stronger temperature dependence than the expected superheating field in the lower reduced temperature range.

The rf critical magnetic field was also studied at 90 MHz for pure tin and indium, and for a series of SnIn and InBi alloys spanning both type I and type II superconductivity. The samples were spherical with typical diameters of 1-2 mm and a helical resonator was used to generate the rf magnetic field in the measurement. The results of pure samples of tin and indium showed that a vortex-like nucleation of the normal phase was responsible for the superconducting-to-normal phase transition in the rf field at temperatures up to about 0.98-0.99 T_c' where the ideal superheating limit was being reached. The results of the alloy samples showed that the attainable rf critical fields near T_c were well described by the superheating field predicted by the one-dimensional GL theory in both the type I and type II regimes. The measurement was also made at 300 MHz resulting in no significant change in the rf critical field. Thus it was inferred that the nucleation time of the normal phase, once the critical field was reached, was small compared with the rf period in this frequency range.

I. Thermochemistry and reaction kinetics of disolvated protons by ion cyclotron resonance spectroscopy. II. Thermochemical studies of small fluorocarbons by photoionization mass spectrometry

The disolvated proton, H(OH₂)₂⁺ is employed as a chemical reagent in low pressure (˂ 10^-5 torr) investigations by ion cyclotron resonance spectroscopy. Since termolecular reactions are absent at low pressure, disolvated protons are not generally observed. However H(OH₂)₂⁺ is produced in a sequence of bimolecular reactions in mixtures containing H₂O and one of a small number of organohalide precursors. Then a series of hydrated Lewis bases is produced by H₃O⁺ transfer from H(OH₂)₂⁺. In Chapter II, the relative stability of hydrated bases containing heteroatoms of both first and second row elements is determined from the preferred direction of H₃O⁺ transfer between BH(OH₂)⁺ complexes. S and P containing bases are shown to bind H₃O⁺ more weakly than O and N bases with comparable proton affinities. A simple model of hydrogen bonding is proposed to account for these observations.

H⁺ transfer from H(OH₂)₂⁺ to several Lewis bases also occurs at low pressure. In Chapter III the relative importance of H₃O⁺ transfer and H⁺ transfer from H(OH₂)₂⁺ to a series of bases is observed to be a function of base strength. Beginning with CH₃COOH, the weakest base for which H⁺ transfer is observed, the importance of H⁺ transfer increases with increasing proton affinity of the acceptor base. The nature of neutral products formed from H(OH₂)₂⁺ by loss of H⁺ is also considered.

Chapters IV and V deal with thermochemistry of small fluorocarbons determined by photoionization mass spectrometry. The enthalpy of formation of CF₂ is considered in Chapter IV. Photoionization of perfluoropropylene, perfluorocyclopropane, and trifluoromethyl benzene yield onsets for ions formed by loss of a CF₂ neutral fragment. Earlier determinations of ΔH^°_f298 (CF₂) are reinterpreted using updated thermochemical values and compared with results of this study. The heat of formation of neutral perfluorocyclopropane is also derived. Finally, the energetics of interconversion of perfluoropropylene and perfluorocyclopropane are considered for both the neutrals and their molecular ions.

In Chapter V the heats of formation of CF₃⁺ and CF₃I⁺are derived from photoionization of CF₃I. These are considered with respect to ion-molecule reactions observed in CF₃I monitored by the techniques of ion cyclotron resonance spectroscopy. Results obtained in previous experiments are also compared.

I. Configurational stability and redistribution equilibria in organomagnesium compounds. II. Redistribution equilibria in organocadmium compounds

I. CONFIGURATIONAL STABILITY AND REDISTRIBUTION EQUILIBRIA IN ORGANOMAGNESIUM COMPOUNDS

The dependence of the rate of inversion of a dialkylmagnesium compound on the solvent has been studied.

Examination of the temperature dependence of the nuclear magnetic resonance spectrum of 1-phenyl-2-propylmagnesium bromide in diethyl ether solution indicates that inversion of configuration at the methylene group of this Grignard reagent occurs with an approximate rate of 2 sec^-1 at room temperature. This is the first example of a rapid inversion rate in a secondary Grignard reagent.

The rates of exchange of alkyl groups between dineopentylmagnesium and di-s-butylmagnesium, bis-(2-methylbutyl)-magnesium and bis-(4, 4-dimethyl-2-pentyl)-magnesium respectively in diethyl ether solution were found to be fast on the nmr time scale. However, the alkyl group exchange rate was found to be slow in a diethyl ether solution of dineopentylmagnesium and bis-(2-methylbutyl)-magnesium containing N, N, N', N'-tetramethylethylenediamine. The unsymmetrical species neopentyl-2-methylbutyl-magnesium was observed at room temperature in the nmr spectrum of the solution containing the diamine.

II. REDISTRIBUTION EQUILIBRIA IN ORGANOCADMIUM COMPOUNDS

The exchange of methyl groups in dimethylcadmium has been studied by nuclear magnetic resonance spectroscopy. Activation parameters for the methyl group exchange have been measured for a neat sample and for a solution in tetrahydrofuran. The exchange is faster in the basic solvent tetrahydrofuran relative to the neat sample and in tetrahydrofuran solution is retarded by the solvating agent N, N, N’, N’-tetramethylethylenediamine and greatly increased by cadmium bromide. The addition of methanol to a solution of dimethylcadmium in tetrahydrofuran appears to have very little effect on the rate of exchange. The exchange was found to proceed with retention of configuration. The rate-limiting step for the exchange of methyl groups in a basic solvent appears to be the dissociation of coordinating solvent from dimethylcadmium.

The equilibrium between methylcadmium bromide, dimethylcadmium and cadmium bromide in tetrahydrofuran solution has also been studied. At room temperature the interconversion of the species is very fast on the nmr time scale but at -100° distinct absorptions for methylcadmium bromide and imethylcadmium are observed.

The species ethylmethylcadmium has been observed in the nmr spectrum.

The rate of exchange of vinyl groups in a solution of divinylcadmium in tetrahydrofuran has been found to be fast on the nmr time scale.

Design, Synthesis, and Study of Novel Platforms for Iron-N2 Chemistry and Photoinduced, Copper-mediated C-N Bond Formation

Several new ligand platforms designed to support iron dinitrogen chemistry have been developed. First, we report Fe complexes of a tris(phosphino)alkyl (CP^iPr₃) ligand featuring an axial carbon donor intended to conceptually model the interstitial carbide atom of the nitrogenase iron-molybdenum cofactor (FeMoco). It is established that in this scaffold, the iron center binds dinitrogen trans to the C_alkyl anchor in three structurally characterized oxidation states. Fe-C_alkyl lengthening is observed upon reduction, reflective of significant ionic character in the Fe-C_alkyl interaction. The anionic (CP^iPr₃)FeN₂^- species can be functionalized by a silyl electrophile to generate (CP^iPr₃)Fe-N₂SiR₃. This species also functions as a modest catalyst for the reduction of N₂ to NH₃. Next, we introduce a new binucleating ligand scaffold that supports an Fe(μ-SAr)Fe diiron subunit that coordinates dinitrogen (N₂-Fe(μ-SAr)Fe-N₂) across at least three oxidation states (Fe^IIFe^II, Fe^IIFe^I, and Fe^IFe^I). Despite the sulfur-rich coordination environment of iron in FeMoco, synthetic examples of transition metal model complexes that bind N₂ and also feature sulfur donor ligands remain scarce; these complexes thus represent an unusual series of low-valent diiron complexes featuring thiolate and dinitrogen ligands. The (N₂-Fe(μ-SAr)Fe-N₂) system undergoes reduction of the bound N₂ to produce NH₃ (~50% yield) and can efficiently catalyze the disproportionation of N₂H₄ to NH₃ and N₂. The present scaffold also supports dinitrogen binding concomitant with hydride as a co-ligand. Next, inspired by the importance of secondary-sphere interactions in many metalloenzymes, we present complexes of iron in two new ligand scaffolds ([SiP^NMe₃] and [SiP^iPr₂P^NMe]) that incorporate hydrogen-bond acceptors (tertiary amines) which engage in interactions with nitrogenous substrates bound to the iron center (NH₃ and N₂H₄). Cation binding is also facilitated in anionic Fe(0)-N₂ complexes. While Fe-N₂ complexes of a related ligand ([SiP^iPr₃]) lacking hydrogen-bond acceptors produce a substantial amount of ammonia when treated with acid and reductant, the presence of the pendant amines instead facilitates the formation of metal hydride species.

Additionally, we present the development and mechanistic study of copper-mediated and copper-catalyzed photoinduced C-N bond forming reactions. Irradiation of a copper-amido complex, ((m-tol)₃P)₂Cu(carbazolide), in the presence of aryl halides furnishes N-phenylcarbazole under mild conditions. The mechanism likely proceeds via single-electron transfer from an excited state of the copper complex to the aryl halide, generating an aryl radical. An array of experimental data are consistent with a radical intermediate, including a cyclization/stereochemical investigation and a reactivity study, providing the first substantial experimental support for the viability of a radical pathway for Ullmann C-N bond formation. The copper complex can also be used as a precatalyst for Ullmann C-N couplings. We also disclose further study of catalytic C_alkyl-N couplings using a CuI precatalyst, and discuss the likely role of [Cu(carbazolide)₂]^- and [Cu(carbazolide)₃]^- species as intermediates in these reactions.

Finally, we report a series of four-coordinate, pseudotetrahedral P₃Fe^II-X complexes supported by tris(phosphine)borate ([PhBP₃Fe^R]^-) and phosphiniminato X-type ligands (-N=PR'₃) that in combination tune the spin-crossover behavior of the system. Low-coordinate transition metal complexes such as these that undergo reversible spin-crossover remain rare, and the spin equilibria of these systems have been studied in detail by a suite of spectroscopic techniques.

I. The structure and properties of closed circular duplex DNA. II. Chemically interacting systems at equilibrium in a buoyant density gradient

I. The binding of the intercalating dye ethidium bromide to closed circular SV 40 DNA causes an unwinding of the duplex structure and a simultaneous and quantitatively equivalent unwinding of the superhelices. The buoyant densities and sedimentation velocities of both intact (I) and singly nicked (II) SV 40 DNAs were measured as a function of free dye concentration. The buoyant density data were used to determine the binding isotherms over a dye concentration range extending from 0 to 600 µg/m1 in 5.8 M CsCl. At high dye concentrations all of the binding sites in II, but not in I, are saturated. At free dye concentrations less than 5.4 µg/ml, I has a greater affinity for dye than II. At a critical amount of dye bound I and II have equal affinities, and at higher dye concentration I has a lower affinity than II. The number of superhelical turns, τ, present in I is calculated at each dye concentration using Fuller and Waring's (1964) estimate of the angle of duplex unwinding per intercalation. The results reveal that SV 40 DNA I contains about -13 superhelical turns in concentrated salt solutions.

The free energy of superhelix formation is calculated as a function of τ from a consideration of the effect of the superhelical turns upon the binding isotherm of ethidium bromide to SV 40 DNA I. The value of the free energy is about 100 kcal/mole DNA in the native molecule. The free energy estimates are used to calculate the pitch and radius of the superhelix as a function of the number of superhelical turns. The pitch and radius of the native I superhelix are 430 Å and 135 Å, respectively.

A buoyant density method for the isolation and detection of closed circular DNA is described. The method is based upon the reduced binding of the intercalating dye, ethidium bromide, by closed circular DNA. In an application of this method it is found that HeLa cells contain in addition to closed circular mitochondrial DNA of mean length 4.81 microns, a heterogeneous group of smaller DNA molecules which vary in size from 0.2 to 3.5 microns and a paucidisperse group of multiples of the mitochondrial length.

II. The general theory is presented for the sedimentation equilibrium of a macromolecule in a concentrated binary solvent in the presence of an additional reacting small molecule. Equations are derived for the calculation of the buoyant density of the complex and for the determination of the binding isotherm of the reagent to the macrospecies. The standard buoyant density, a thermodynamic function, is defined and the density gradients which characterize the four component system are derived. The theory is applied to the specific cases of the binding of ethidium bromide to SV 40 DNA and of the binding of mercury and silver to DNA.