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The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. The first comprehensive measurements of the temperature-dependent NO rebinding kinetics of Mb and its distal pocket mutants are presented.

Hydration reactions of fully oxidized alumina clusters are measured with a quadrupole ion trap secondary ion mass spectrometer (IT-SIMS) 68-71, wherein the number of Lewis acid sites is determined. The observations set the stage for reactivity studies of the alumina clusters, which has the potential for close resemblance to hydroxylated surfaces.

The direct detection of the nerve agent VX (methylphosphonothioic acid, S-[2-[bis(1-methylethyl)aminolethyl] Oethyl ester) on milligram quantities of soil particles has been achieved using ion trap secondary ion mass spectrometry (IT-SIMS). VX is highly adsorptive toward a wide variety of surfaces; this attribute makes detection using gas-phase approaches difficult but renders the compound very amenable to surface detection. An ion trap mass spectrometer, modified to perform SIMS, was employed in the present study. A primary ion beam (Re[[O.sub.4].sup.-]) was fired on axis through the ion trap, where it impacted the soil particle samples. [VX + H]-, [[VX + H].sup.+] fragment ions, and ions from the chemical background were sputtered into the gas-phase environment of the ion trap, where they were either scanned out or isolated and fragmented (M[S.sup.2]). At a surface concentration of 0.4 monolayer, intact [[VX + H].sup.-], and its fragment ions, were readily observable above background. However, at lower concentrations, the secondary ion signal from VX became obscured by ions derived from the chemical background on the surface of the soil particles. M[S.sup.2] analysis using the ion trap was employed to improve detection of lower concentrations of VX: detection of the 34S isotopic ion of [[VX + H].sup.+], present at a surface concentration of [approximately]0.002 monolayer, was accomplished. The study afforded the opportunity to investigate the fragmentation chemistry of VX. Semiempirical calculations suggest strongly that the molecule is protonated at the N atom. Deuterium labeling showed that formation of the base peak ion ([C.sub.2][H.sub.4])N[[(i-[C.sub.3][H.sub.7]).sub.2].sup.+] involves transfer of the amino proton to the phosphonothioate moiety prior to, or concurrent with, C-S bond cleavage. To manage the risk associated with working with the compound, the vacuum unit of the IT-SIMS was located in a hood, connected by cables to the externally located electronics and computer.

A method for the identification of mineral phases on basalt surfaces utilizing secondary ion mass spectrometry (SIMS) with imaging capability is described. The goal of this work is to establish the use of imaging SIMS for characterization of the surface of basalt. The basalt surfaces were examined by interrogating the intact basalt (heterogeneous mix of mineral phases) as well as mineral phases that have been separated from the basalt samples. Mineral separates from the basalt were used to establish reference spectra for the specific mineral phases. Electron microprobe and X-ray photoelectron spectroscopy were used as supplemental techniques for providing additional characterization of the basalt. Mineral phases that make up the composition of the basalt were identified from single-ion images which were statistically grouped. The statistical grouping is performed by utilizing a program that employs a generalized learning vector quantization technique. Identification of the mineral phases on the basalt surface is achieved by comparing the mass spectra from the statistically grouped regions of the basalt to the mass spectral results from the mineral separates. The results of this work illustrate the potential for using imaging SIMS to study adsorption chemistry at the top surface of heterogeneous mineral samples.

The FixL heme-based sensor is more reactive than mioglobin to the large polar ligand imidazole. The binding of this ligand to the FixL heme domain has been measured to determine which features of a myoglobin heme favor binding of imidazole. Apart from histidine, association rate constants fell more than 3000-fold as the volume of the E7 side chain was expanded from alanine to phenylalanine. Imidazole binding inhibition was due to displacement of coordinated water from H64 and H64Q.

A study was conducted to analyze the unfolding behavior of different distal heme pocket mutants of sperm whale deoxymyoglobin. Kinetic data were observed by monitoring the absorption spectrum after sample injections while the absorption and RR spectra were obtained at ambient temperatures. Results showed that none of the distal heme pocket mutations influenced the nature of the deoxy-heme intermediates formed under low pH unfolding environments.

A study was conducted to analyze a transient open form of the ligand pathway in Tyr64(E7) myoglobin. Time-resolved ultraviolet resonance Raman experiments were performed to examine the mechanism of ligand entry. A probe beam was generated by the H2-first Stokes shift of the third harmonic of an Nd:YAG laser. Experimental results indicated the presence of the transient conformation wherein Tyr64 swings out of the heme pocket and is subjected to solvent.

A study aimed at evaluating static secondary ion mass spectrometry's (SIMS) practical use in pesticide analysis is presented. The SIMS was utilized for the direct surface analyses of pesticides absorbed on dandelion leaves, grass, soil, and stainless steel samples. Direct surface analysis edges out conventional pesticides analysis methods in the elimination of sample pre-treatment including extraction. The results of the study indicate that SIMS can ideally be used in the direct surface detection of a range of pesticides.

The mechanism by which specific amino acid residues regulate the binding of myoglobin to hemin was investigated by measuring the rates of hemin dissociation from approximately 100 different metmyoglobin mutants. Mutants for histidine (His93(F8)) and leucine (Leu89(F4)) showed more than 100-fold increases in the rate of hemin loss at pH 5 and 7, while mutations of Leu29(B10), Phe46(CD4) and Gly65(E8) caused more than 10-fold increases in hemin loss. The differences in the rates of hemin loss resulting from the mutations were attributed to the nature of chemical bonds within the residues.

The role of heme in protein stability was determined by examining the binding activities of heme and globin in wild-type, V68T and H97D sperm whale myoglobins. Measurements of hemin loss and analysis of the unfolding reactions of the different globins showed the association of heme dissociation with heat-induced denaturation of holomyoglobins and hemoglobins, suggesting that heme protects proteins from denaturation.

The role of globin structure in the regulation of the bimolecular association of heme with apoglobins was determined by measuring the association rate constants of the binding interaction of carbon monoxide (CO)-heme to apomyoglobin mutants with substitutions in the heme pocket. The formation of heme-protein complexes was associated with slow reaction rates, with an overall association rate of approximately 1 x 10(super14) M-1.

An analysis of residues vital for the cell-proliferative activity of proteins reveals that there are two receptor binding sites on the surface of the human granulocyte colony-stimulating factor (G-CSF). This fact validates the dimerization model for receptor activation and indicates that this mechanism can be used for cytokine signal transduction. Most of the residues mutated to alanine remain fully active biologically, but some of the variants are unable to stimulate the in vitro proliferation of bone marrow cells. Alanine scanning mutagenesis of G-CSF is used for the analysis.

The mechanism of hydrogen cyanide binding to mammalian myoglobin was investigated using kinetic measurements of cyanide dissociation from four sets of mutants under physiological conditions. Data showed that bond formation and mutation within the distal pocket of the heme protein which affects its polarity determined the efficiency of cyanide binding to myoglobin. The ease of water displacement from the ferric atom, electrostatic interactions at the sixth coordination position and steric hindrance also affected the rate of cyanide dissociation.

Binding of nitric oxide (NO) to oxyhemoglobin (HbO2) has been suggested as the major cause of NO depletion which leads to mild hypertensive events. Herein, the kinetics of NO-induced oxidation of recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb) were measured using rapid mixing techniques to understand its mechanism. The rate of oxidation of MbO2 and HbO2 was dependent on NO concentration but not on oxygen concentration. The process occurred in the distal heme pocket and formed Fe3+-OH2 and nitrate as end products.

The relationship between the distribution of the unpaired electron in P+ and the circular dichroism (CD) anisotropy of P+ has been determined. CD anisotropy values were measured for the far-red absorption band of P+ in reaction centers of the purple bacteria, Rhodopseudomonas viridis and Rhodobacter sphaeroides, and one green sulfur bacterium, Chlorobium tepidum. Different CD anisotropy values were obtained for P960+, P870+ and P840+. The results also showed different configurations between the differen P+.

Site-directed mutations to study the role of important distal pocket residues and to mimic the heme ambience of Aplysia limacina myoglobin which attains high O2 affinity in the absence of a distal histidine indicates that it is impossible to adapt the mammalian myoglobins to identify ligands in the manner of Aplysia myoglobin with a small number of substitutions in the heme pocket. The impact of second-site mutations in the valine environment in several cases are contrary to those observed for the corresponding mutations in the wild type environment.

The D-helix is necessary in hemoglobin beta subunit and myoglobin for inhibition of autooxidation and hemin loss indicating that the rate of hemin loss and confirmation of the CD loop can be affected by Met(super 55). The alpha hemoglobin subunit does not contain the D-helix because of the deletion of the five consecutive residues whereas this deletion of D-helix in beta subunit in human hemoglobin enhances the rate of hemin loss. The denaturation of myoglobin and hemoglobin takes place in three steps.

Examination of the kinetics of hemin loss and equilibrium unfolding parameters for 22 mutant apoglobins with replacements at the distal portion of the heme pocket helps analyze various factors controlling the stability of sperm whale, pig and human metmyoglobin. Unstable globin structures exhibit no correlation with hemin loss kinetics and polar residues in the distal pocket decrease the overall stability of apomyoglobin. Both apoprotein folding and binding of the prosthetic group influence the stability of heme proteins.

Two efficient chiral synthetic techniques help synthesize multigram quantities of threo-3-hydroxylysine, a naturally occurring amino acid and an intermediate in the synthesis of balanol, a potent protein kinase C inhibitor. One technique uses Hayashi's chiral ferroceno gold catalyst to synthesize (2R, 3S)-3-hydroxylysine and the other technique uses Sharpless chiral cis-hydroxylation to prepare beta-hydroxy-alpha-amino acids.

Analysis of the mechanisms of selective discrimination of ligand binding in hemoglobin and myoglobin involves site-directed mutagenesis and physical and structural characterizations. Myoglobin is a compact, alpha-helical globular protein that stores oxygen during periods of rest until required for phosphorylation. Distal histidine mediates stabilization of binding to hydrogen and oxygen and inhibits heme iron autoxidation by a preferential binding of O(sub 2) to CO.

Infrared spectra and CO binding analysis of 41 recombinant myoglobins reveals that the myoglobin bound CO vibrational spectrum is an accurate gauge of ambient electrostatic potentials. Steric hindrance does not affect Fe-C-O complex bond orders and geometry. The CO-myoglobin IR spectrum substrates reflect the binding site electrostatic potential.

X-ray crystallography and infrared spectroscopy were used to achieve 2.1- and 2.2 amstrong resolution in the crystal structures of the deoxy and carbonmonoxy versions of a digtal pocket myoglobin mutant, in which threonine replaces valine. The monocoordinated water molecular in the distal pocket can develop hydrogen bond with the N(epsilon)-H of the distal histidine ared O(gamma) of threonine, without effecting changes in the protein structure.

The effect of apolar substitution at E11 in the distal heme pocket of the beta-chains of hemoglobin was investigated by replacing Val(super 67)E11 with Thr. The mutation is isosteric and only modifies the polarity of the beta-heme environment. Equilibrium and kinetic measurements of oxygen binding to the mutant indicate a 2-fold decrease in the O2 affinity of human hemoglobin. This effect is due to modifications of the properties of the intermediates of oxygenation and not to changes in the ligand binding parameters of the T- and R-state.

The ligand binding experiments with ferrous and ferric Nostoc sp. (Ns) H-NOX has shown major functional differences between Ns H-NOX and soluble guanylyl cyclase (sGC). The large conformational transition after oxidation has shown that Ns H-NOX can function as either a redox or a NO sensor in the cyanobacterium.

GuHCl-induced, equilibrium unfolding of five sperm whale metMb variants are measured to understand the discrepancies during the unfolding of wild-type holomyoglobin in the ferric state (metMb). The high affinities of native apoMb (N) for hemin are found to have significant influence on the stability of holo-metMb and the partially unfolded intermediate with hemin bound ((IH) exhibiting a hemichrome spectra are indicative of a bis-histidyl axial coordination which is seen clearly when the stability of the N state or its affinity for hemin is reduced.

The pathways identified by alkyl isocyanides (CNRs) for diatomic ligand movement into and out of Mb, with their side chains acting as transition state analogues are reviewed. The strong correlation observed between the fraction of in conformer, [F.sub.in], for long-chain MbCNR complexes, the fraction of geminate recombination of photodissociated [O.sub.2], and the bimolecular rates of [O.sub.2] entry into the distal pocket revealed that alkyl isocyanides serve as transition state analogues for the movement of [O.sub.2] into and out of the binding pocket of Mb.

A study is conducted to gain insight into the low- and high-frequency peaks observed for the alkyl isocyanides upon binding to sperm whale myoglobin (Mb). The internal rebinding for in alkyl side chain conformation is promoted by the photodissociated ligand which is stuck in the distal pocket while the out conformation that inhibited geminate recombination is attributed to the part of the ligand that is already in an open E7 channel, poised for rapid escape.

Two major conformations, the in conformer, His(E7) in a closed position and the out conformer His(E7) in an open position observed for the crystal structures of methyl, ethyl, propyl, and butyl isocyanide bound to sperm whale myoglobin (Mb) are reported. The unfavorable hindrance caused with crowding a large alkyl side chain into the distal pocket is found to be roughly equal to that for pushing open the His(E7) gate and is easily affected by crystal packing.

Visible and ultraviolet resonance Raman (RR) spectra are described for [Fe.sup.III](NO) adducts of myoglobin variants with altered polarity in the distal heme distals. The implications are discussed for biologically important processes, which have included NO binding, reductive nitrosylation and NO reduction.

The studies have shown that water is detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not observed in the corresponding crystal structures. The spectral perturbation of pentacoordinate heme has offered a robust system for measuring nonspecific hydration of the active sites of heme proteins.

Flash photolysis studies of ([alpha]-cp[beta] ) were performed to understand the biphasic CO and O2 kinetics for a circularly permuted human [beta]-globin (cp[beta]) coexpressed with human [alpha]-globin in bacterial cells which form [alpha]-cp[beta] hemoglobin in solution. The equilibrium oxygen binding measurements obtained for [alpha]-cp[beta] supported the generated intermediate, higher affinity, deoxyHb quaternary state by the deoxygenated permutated [beta] subunits.

The various effects of the interfacial and distal-heme pocket mutations on the structure, as well as the functions of hemoglobin and hemoglobin-based oxygen carriers are discussed. The different combinations of these mutations are shown to lead to the formation of hemoglobin molecules with excellent ligand-binding and allosteric properties.

Complexes containing the U(V) cation [UO2](super +) would add molecular oxygen in the gas phase and the reactive motif of the U(V) species [UO2](super +) is that of a Lewis acid accepting electron density from donor ligands. Some ligand complexes of [UO2](super +) are shown to add molecular dioxygen, indicating donation of electron density from the metal center.

A study was conducted to measure the CO stretching frequencies for a comprehensive set of distal pocket mutants of Lba. The results suggest that there is a direct interaction between the His(super E7) and Tyr(super B10) side chains in Lba that weakens stabilization of bound ligands.

Ion trap-secondary ion mass spectrometer is used to study the formation of adduct ions consisting of uranium oxycations and water. It is indicated that stability of the hydrate complexes [UOyHz(H2O)(sub n)](super +) increases with increasing n, until the optimum number of ligands is achieved.

Research indicates that in heme proteins the metal-ligand vibrational frequencies are complex for NO-heme adducts vis a vis their CO-heme counterparts. Furthermore, extra antibonding electron on NO introduce additional complexity. Data indicate that the vibrational frequencies are sensitive to electronic polarization and bond strength to the trans axial ligand.

Valine(supra-68) was replaced by asparagine in an attempt to engineer higher oxygen affinity to investigate the role of polar interactions in the distal heme pocket. The substitution led to an increase in oxygen affinity and a decrease in carbon monoxide affinity. Functional and structural results indicated that the asparagine side chain forms a strong hydrogen bond with bound oxygen through its -NH2 group but blocks the approach of ligands to the iron from within the distal heme pocket.

The effects of mutating His64 and Val68 on the CO infrared stretching bands associated with the B1 and B2 photoproduct states are reexamined. Fourier transform infrared (FTIR) spectroscopy and density functional theory (DFT) calculations were combined to examine the effects of the electrostatic environment on the B state photoproduct bands.

The Fe-CO unit in heme proteins has acted as a sensitive structural probe of the heme active site. Detailed information about protein-ligand interactions were obtained through measurement of C-O stretching (vC-o), Fe-CO stretching (vFe-Co) and Fe-C-O bending (delta FeCO) frequencies. RR spectroscopic evidence indicates a hydrogen bond in the A1 conformation of MbCO, implying that a proton exists and contributes to the raised vFE-CO and decreased vC-o.

Analysis of the Fe-CO stretching frequencies of oxymyoglobin revealed chemical variations between the Fe-O2 and Fe-CO adducts. The globin residues affected the bond strength and vibrational properties of the oxymyoglobin complex. The highly polar Fegamma+-O-Ogamma- systems have fixed bond orders with no alternative resonance configurations. The apolar Fe-C-O system is an admixture of two alternative resonance structures with net changes in bond order.

The peaks of the infrared spectrum of carbon monoxide bound to myoglobin can be seen as a sensitive measure of electrostatic fields near the ligand binding site. Electrostatic analyses of ligand binding and Vc-o seem to be applicable in general to heme proteins.

Interactions of nitric oxide with a heme in proteins have become a particularly interesting area of research. A study therefore investigated the resonance Raman spectra of nitric oxide adducts of mutated myoglobin proteins with particular attention to the Fe-histidine stretching and N-O stretching mode frequency bands to elucidate the resonance Raman spectra of the NO adduct. Experimental materials, methods, results and conclusions are discussed.

The kinetics of ligand binding to cyanoglobin is explored to clarify the mechanistic origin of its high oxygen affinity. Through a comparison of the physiological properties of cyanoglobin, leghemoglobin a, and sperm whale myoglobin, a site-directed mutagenesis analysis is conducted. Results provide support for a model of cyanoglobin function proposed by Hill et al. In this model, cyanoglobin sequesters oxygen and presents it to a terminal cytochrome oxidase complex under microaerobic conditions, when nitrogen fixation and ATP demand is maximal.