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The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.

The synthesis, characterization, and electrochemical studies of [beta],[beta]'-fused metallocenoporphyrins are described. The novel Pd(0) catalyzed [3@] cycloaddition reaction with porphyrins was studied, a series of [beta][beta]' fused monoruthenoceno metalloporphyrins and bismetalloporphyrins with different central metals were prepared and significant electronic interactions between the metallocene and metalloporphyrin were observed.

A study was conducted to investigate in detail the [super 1]H NMR spectra of the ferriheme center and nearby protein residues in the high- and low-spin forms of the nitrophorins (NPs) from Rhodnius prolixus. The structures of the ferriheme complexes in terms of the orientation of the histidine imidazole ligand orientations depends on the heme cavity shape and could be altered by mutation of amino acids with side chains (phenyl vs tyrosyl) near the potential position where a heme vinyl group would be in one of the isomers.

The hydrogen bonding of ligated water in ferric, high-spin, resting-state substrate complexes of heme oxygenase from Neisseria meningitides is systematically perturbed by variable electron-withdrawing substituents on the hemin periphery. The pattern of [sup.1.H] NMR-detected dipolar shifts due to the paramagnetic anisotropy is strongly conserved among the four complexes, with the magnitude of dipolar shifts or anisostropy increasing in the order of substituent formyl

The use of mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc(sub 1) as a starting point for the design of a water-soluble model for this protein is presented. NMR spectra reveal that the protein exists in solution in a unique conformation and is guided to structural studies.

Solution (Super 1)H NMR was used to elucidate the magnetic properties and electronic structure of the prosthetic group in high-spin, ferrous deoxy myoglobin from the sea hare Aplysia limacina. Understanding of the NMR spectra of high-spin ferrous hemoproteins requires the determination of the anisotropy and orientation of the paramagnetic susceptibility tensor from a variety of proteins with varied active site geometry.

Research has been conducted on nickel(II) porphyrins. The electrochemistry of these compounds has been investigated in the effort to understand the effect of structural and electronic factors on electron-transfer processes in porphyrins, and the details are reported.

Research describes synthesis of a new series of heterobimetallic cofacial diporphyrins containing luminescent Ga(III) centers. The reduction of dioxygen by cofacial cobalt(II) diporphyrins is studied by photophysical and spectroscopic methods.

Research has been conducted on the reaction of porphyrins with osmium tetraoxide. The effect of the electron-donating and electron-withdrawing substituents on these porphyrins has been investigated.

The preparation and investigation of a novel trimeric porphyrin array with directly fused macrocycles via their beta-pyrrolic carbons, is described.

Research has been conducted on the dissociation of PH (sub)3 from the 18-electron system CpMoX(PH (sub)3) (sub)3. The use of the density functional theory is examined.

A study was conducted to analyze the cyanomet myoglobin complexes od different chemically altered hemins to determine the importance of hemin peripheral electronic in increasing the orbital degeneracy of the pi-bonding orbitals. 1H nuclear magnetic resonance spectroscopy was utilized to carry out the analysis. Results indicated that the peripheral substitution of the hemin can change the rhombic splitting of the orbitals in a predictable way.

A study was conducted to examine the effects of the symmetry of substitution pattern and substituent orientations on the out-plane and in-plane distortions of the macrocyle of nickel porphyrins. These nickel porphyrins were analyzed through the use of molecular mechanics calculations, X-ray crystallography, UV visible absorption spectroscopy and resonance Raman spectroscopy. Results revealed that out-plane and in-plane distortions were dependent on perturbation symmetry of the peripheral substituents.

The excited-state lifetimes and fluorescence of the nickel(II) analogue, NiT(t-Bu)P, and its similar complex, NiT(Ad)P, were studied. The temperature reaction was distinct among nonplanar zinc and free base porphyrins even at the distorted ground state. It was revealed that the lifetime of the analogue's excited state was different from several picoseconds to tens of nanoseconds. The excited-state relaxation mechanics relied on solvent dielectric properties and temperature. Nonplanar distortions are relevant to the photophysical properties of NiT(t-Bu)P.

Research was conducted to study the influence of central metals of various sizes on the macrocyclic structure of a series of 5,15-disubstituted metallophorphyrins. Methodologies used included X-ray crystallography, resonance Raman spectroscopy and molecular mechanics. The contributions of the ruf and dom deformations in the crystal structures of several metal complexes of the phorphyrins were found to be metal-dependent. Results indicate the quantitative validity of normal-coordinate structural decomposition.

A novel protection/deprotection technique for the synthesis of 8-de-ethyl-8-vinylchlorophyll-a is presented. The procedure involves regioselective osmylation on ring-B and subsequent dehydration of the resulting 7,8-diol. Three synthetic routes starting from chlorophyll-a to 8-de-ethyl-8-vinyl-chlorin-e(sub 6) trimethyl ester are developed. The first syntheses of 8-de-ethyl-8-vinyl derivatives of chlorophyll-a are also reported.

The synthesis and characterization of a series of monometallo-, bimetallo-, and heterobimetallo-1,2-ethene-linked cofacial bisporphyrins are presented. Two heterobimetallic cofacial bisporphyrins were synthesized easily by initially forming monometalated species with subsequent insertion of a second metal ion. Initial monometalations were observed to be slower under dilute conditions in CH2Cl2/MeOH at refluc but under dilute conditions in hot DMF, the insertion rate was sufficiently fast to enable isolation of the monometalated cofacial isomers as the major product.

Solution 1D and 2D NMR, together with limited isotope labeling, have led to the assignment of the heme, axial His, and numerous heme contact residues in sperm whale, horse, and human deoxy myoglobin. The paramagnetic relaxivity leads to increased line widths and shorter [T.sub.1]s with little compensation in increased dispersion due to dipolar shifts. Hence only limited, but crucial F helix standard backbone sequence specific assignment could be made for heme cavity residues. Numerous other residues with significant dipolar shifts could be assigned from the characteristic scalar connectivities and dipolar contacts to the heme predicted by the crystal structure. It is concluded that the complete and unambiguous assignment of the heme pyrrole substituent signals is not attainable by 2D NMR alone without either partial deuterium labeling of the heme or parallel assignment of key residues in dipolar contact with the heme; hence the present study revises some earlier assignments. The resulting dipolar shifts for nonligated residues, together with the crystal coordinates of deoxy myoglobin, were used to determine the orientation relative to the heme and the anisotropy of the paramagnetic susceptibility tensor. The significant anisotropy, [absolute value of [Delta][Chi]] [similar to] 1 x [10.sup.-9] [m.sup.3]/mol, however, is shown to result in dipolar shift with reciprocal square, rather than just reciprocal, absolute temperature dependence, which is indicative of large zero field splitting rather than g-tensor anisotropy. The appropriate equation for a 5[B.sub.2] ground state allows an estimate of the zero-field splitting, D [similar to] -10 [cm.sup.-1] which is in good agreement with earlier results. The present NMR data favor a spin-only magnetic moment with S = 2 and D [similar to] -10 [cm.sup.-1] over a ground state with S < 2 and significant orbital contribution (Hendrich and Debrunner, 1989).

An experiment was conducted to examine the electrochemistry and ozonation reactions of manganese (III) dodecaphenylporphyrins compounds and their derivatives. Nuclear magnetic resonance (NMR) and UV-visible spectra were performed on the compounds using UV and NMR spectroscopy. Results showed that each (P)MnCl derivative can exist in five different oxidation states and that all compounds are stable in their neutral form and even during the addition of three electrons.

Methylpheophorbide-a (a chlorophyll a analog) was converted into methyl [13.sup.1]-deoxypyropheophorbide-a 2 in excellent yield. The Ni(II) complex of 2 on hydrogenation and subsequent Viismeier reaction with phosphorus oxychloride and 3-(N,N-dimethylamino)acrolein produced Ni(II) methyl 20-(2-formylvinyl)-[13.sup.1]-deoxypyropheophorbide-a 4. In contrast to other synthetic and naturally occurring isobacteriochlorins, the isobacteriochlorin obtained by acid-catalyzed cyclization of 4 was found to be unstable and oxidized to the corresponding benzochlorin. The free base benzochlorin 7, obtained by demetalation, up on DDQ/methanol treatment produced a mixture of [13.sup.2]-oxo- and [13.sup.1]-methoxy-[13.sup.2]-oxobenzochlorins 8, 9. Surprisingly, under similar reaction conditions, an ethanolic solution of DDQ afforded [13.sup.1], [13.sup.2]-dioxobenzochlorin 13 as a major product. Vilsmeier reaction ([POC1.sub.3]/DMF) of Ni(II) benzochlorin 6 gave unexpected 5-formyl-[13.sup.2]-oxobenzochlorin 15. Zn(II) complexes of the newly synthesized benzochlorins showed long wavelength absorptions in the range of 750-753 nm. Thus, compared to the respective free base analogues bathochromic shifts of 4042 nm were observed. Despite extensive studies in benzochlorins, these are the first examples which exhibit such remarkable long wavelength absorptions in their electronic absorption spectra. The structures of novel benzochlorins were confirmed by extensive NMR [2D NMR (ROESY), 13C NMR] and X-ray crystallographic studies. On the basis of the crystal structure of oxobenzochlorin 8, the chemical reactivity of other benzochlorin analogues were examined by semiempirical molecular orbital theory. Our results are in agreement with the previous qualitative electron sextet hypothesis proposed for chlorin systems by Woodward. The fluorescence quantum yields and the singlet oxygen yields of the free base and Zn(II) benzochlorins were measured relative to tetraphenylporphyrin (TPP) in benzene.

Corrole is a tetraphrrolic macrocycle present in octamethylcorrole. It can stabilize higher oxidation states for coordinated metals and its molecular skeleton can maintain a planar conformation. The unusual reactivity of corrole in the Vilsmeier reaction is reported. Also a direct synthesis of formylcorroles by prefunctionalized a,c-biladiene salts is described.

A novel methodology of synthesizing pentapyrrolic expanded porphyrins, sapphyrins, was developed. The approach was characterized by acid-catalyzed condensation of 1,19-diunsubstituted a,c-biladienes and 3,4-dialkylpyrrole-2-carbaldehydes which eliminated the preparation of bipyrrolic intermediates. The approach also involved the preparation of sapphyrins with symmetrical distribution of the peripheral substituents.

Copper(II) oxidation procedure was utilized to cyclize a,c-biladienes containing 1- and/or 19-(para-tolylmethyl) substituents yielding numerous products wherein 1- or 19-substituents had modified positions in the tetrapyrrole macrocycle. a,c-Biladiene coordinates rendered copper(II) 1-(para-tolylmethyl)-20-para-tolyl macrocyclic compounds, copper(II) 2-methylidenechlorin and copper(II) porphyrins upon copper(II)-assisted cyclization. para-Tolyl-methyl group displayed more stable contributions of methylene both as macrocyclic bridging carbon and interpyrrolic carbon atom.

Anionic and paramagnetic [CpCr(NO)X2]- complexes were synthesized by the reaction of [NBu4]I with [CpCr(NO)I]2. The CpCr(NO)X2 anions exhibited single strong nitrosyl-stretching absorption that was generated by a linear nitrosyl group. The formation of CpCr(NO)Cl2 from the oxidation of [CpCr(NO)Cl2]- also involved the formation of an initial oxidation product that was unstable due to the loss of the NO unit. Furthermore, the stability of the anionic and paramagnetic complexes were dependent on pi-donation from alkoxide or amide ligands which reinforced the low-spin state.

X-ray crystallography and proton nuclear magnetic resonance spectroscopy were done to determine the structure of symmetrically and asymmetrically highly substituted porphyrins. The results showed that the decasubstituted free-base porphyrins with two meso-aryl substituents have a negligible out-of-plane distortion but a large amount of in-plane distortion, with the corresponding nickel(II) complexes having either planar or modestly nonplanar arrangements. The nickel(II) complexes of 5,15-diethyl-substituted porphyrins, on the other hand, were found to have very ruffled conformations, in spite of the planar arrangement of the corresponding free-base porphyrins.

The regioselective oxidation of pyrrole units in porphyrin and chlorin systems by OsO4 were analyzed to determine the kinetic properties of the synthetic process. Analysis of the OsO4-mediated regioselective oxidation of porphyrin and chlorin systems indicated the role of electron-withdrawing groups at various points of the macrocycles in modulating the synthetic reaction. Furthermore, the position and number of electron-withdrawing groups at various points of the substituents also affected their migratory behavior during pinacol-pinacolone rearrangements.

The effect of substituents with increasing steric demands on the structure of nickel(II) 5,15-disubstituted porphyrins was studied by X-ray crystallography, UV-visible absorption spectroscopy, resonance Raman spectroscopy, molecular energy optimization computations and INDO/s molecular orbital computations. Findings showed that the distortion of the macrocyle for the disubstituted nickel porphyrins is a combination of ruffling and doming distortions. Also, the gal distortion can be precisely described in terms of displacements along the lowest-frequency normal coordinates of B1u and A2u symmetry.

The resonance Raman (RR) spectra of myoglobin reconstituted with seven heme isotopomers was assigned to determine how the oxidation and ligation states of myoglobin affect the structure of its noncovalently bound protoheme group. The assignments were facilitated by labeling the hemes with 15N and meso-D4 at the site of the vinyl and propionate substituents or in the porphyrin backbone. The results demonstrated protein-specific effects via the activation of several out-of-plane modesin the low frequency band of the RR spectra.

The preparation and characterization of 1,2-and 1,1-bis(2-pyrrolyl)ethenes were performed. Trans-1,2-bis(2-pyrrolyl)ethenes were synthesized via the reductive coupling of 2-formylpyrroles under McMurry conditions. In contrast, isomeric 1,1-bis(2-pyrrolyl)ethenes were obtained as a minor byproduct in the Friedel-Crafts reaction between 2-unsubstituted pyrroles and acetic anhydride. In addition, both ethenes were obtained from base-catalyzed elimination of HCl from 5-(chloromethyl)dipyrromethanes produced by reacting 2-unsubstituted pyrroles with chloroacetaldehyde diethyl acetal.

Methods for the synthesis of biscorrole, porphyrin-corrole, bisporphyrin, bischlorin and porphyrin-chlorin heterodimers are presented. The development of the techniques for the synthesis of systems with the corrole nucleaus is particularly challenging since little is known about the synthetic chemistry of corrole. The synthesized products are characterized using a combination of nuclear magnetic resonance spectroscopy, ultraviolet spectrometry, mass spectroscopy and elemental analysis techniques.

Several protoheme variants were reconstituted with peptides that constitute the cytochrome b maquette to test the effects of the heme on the structural and electrochemical properties of the protein. The protoheme and heme variants provides the unequivalent binding properties of the hemes in the dimers by providing opposing rotation of the heme that lends to the character of the dimer. When interactions cannot be mitigated by the rotation of the heme, protein reconfiguration occurs to shift heme spin to a higher configuration.

The synthesis of regiochemically pure porphyrins with D2h symmetry has been described. The scheme involves the tetramerization reactions of two different pyrrole components, 2,5-diunsubstituted pyrrole and 2,5-bis( N,N-dimethylamino methyl)pyrrole. The method has also been applied to the synthesis of unsymmetrical porphyrins with C2v symmetry by the reaction of a tripyrrane and 2,5-bis( N,N-dimethylamino methyl)pyrrole. The structures of the porphyrins have been determined by single-crystal X-ray crystallography.

The bis-imidazole complexes of the vinylhemins 2-vinyldeuterohemin IX, 4-vinyldeuterohemin IX, protohemin IX, and protohemin III were characterized via local density functional calculations and Soret-excitation resonance Raman spectroscopy. Both methods, which shed new light on factors that affect the vibrational behavior of protoporphyrins in the V(sub c=c) mode, revealed the presence of two vinyl torsional conformers of nearly equal energy.

Proton NMR spectroscopic and molecular mechanic studies of seven dodecasubstituted porphyrins reveal that these compounds possess identical structures in both solution and solid states, implying the presence of cavities in solution also. The compounds with these cavities react with 1,3,5-trinitrobenzene to result in pi-complexes and find application as oxidation catalysts with good optical selectivity. The presence of the cavities modifies the physical and chemical properties of the porphyrins.

A single-step reductive coupling of metal(II) 5-formyloctaethylporphyrins produces both cis-1,2- and trans-1,2-ethene-bridged bisporphyrins. Pi-pi aggregation of the monomeric subunits is significant in the formation of cis-ethene-bridged bisporphyrins. Crystal structures of the cis-dimer exhibit both intra- and intermolecular aggregation and the cis-dimers having cofacial bisporphyrins have significant overlap of the pi-system. X-ray and spectroscopic studies help determine the molecular structures of 1, 1-carbinol-bridged bisporphyrins and the trans-1,2-ethene-bridged bisporphyrin.

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic experiments help characterize iron methylchlorin- and mesochlorin-reconstituted myoglobin, horseradish peroxidase and cytochrome b5. A comparative study of these spectra with those of corresponding iron octaethylchlorin complexes helps derive spectral signatures for histidine-ligated iron chlorin-containing proteins. MCD spectroscopy is an efficient technique to determine iron chlorin coordination structure, spin state and oxidation state, and to differentiate two types of green heme systems, iron-chlorins and iron formyl-substituted porphyrins.

X-ray studies help determine the crystal structures of Cu(II) octaethyltetraphenyl porphyrin (OTEPP) and its pi cation radical. Oxidation of Cu(II)OETPP stimulates further deformations of the porphyrin skeleton and the product radical exhibits more distortion than the parent compound. Optical and IR spectral data of cation species are similar to that of the Pi cation radical. NMR spectrum reveals the species as diamagnetic. Spectral data reveal the antiferromagnetic coupling between Cu(II) and the nonplanar pi radical spins in the cation radical.

MCD spectroscopic studies of the electronic structures and coordination behaviors of three nickel porphyrins that have different patterns and amounts of macrocycle deformations reveals A-term peaks for all three compounds, indicating that both ruffled and saddle-shaped conformations retain D(2d) symmetry. MCD spectra of Ni(II) protoporphyrin IX-reconstituted hemoglobin ((Ni)Hb) and myoglobin ((Ni)Mb) reveal that protein does not influence the energy level of nickel porphyrins.

X-ray studies of 3,5,7,13,15,17-hexaethyl-2,8,12,18-tetramethylporphyrin (H2DAP) crystals reveal an elongated planar macrocycle along the 5,15 axis that cancels the steric interference of the meso and pyrrole ethyl groups. Another decaalkylporphyrin NiDAP, reveals nonplanar distortion in the porphyrin unit to relieve steric crowding. Optical spectroscopy, NMR and MO calculations help examine solution conformations of H2DAP and NiDAP. Low-temperature NMR studies reveal cis and trans conformation of the methyl groups of the meso ethyls in H2DAP and ZnDAP.

Time-resolved and steady-state optical analyses of a series of planar and nonplanar metal-free porphyrins reveal the influence of structural perturbations on the electronic characteristics of the porphyrin system. Optical data reveal the mechanisms of excited-state dynamics and major decay routes induced by structural parameters. Nonplanar porphyrins show large Stokes shifts, decreased yields and smaller excited singlet state lifetimes.

H NMR spectroscopic studies on the electronic and functional properties of Asp235 in yeast cytochrome c peroxidase reveal that the spin state and coordination characteristics of the heme iron in cytochrome C peroxidase do not control the substrate oxidation rate. The iron segment influences the pH-based properties of the active site of Asp235Ala variant. Asp235 is vital for the catalytic activity of the enzyme as it regulates exact Trp 191 orientation for electron exchange within cytochrome c peroxidase - cytochrome C complex.

Electronic and steric influences of protohemin IX matrix on substrate-bound heme oxygenase (HO) orientations indicate heterogeneous, pH-dependent kinetics and unchanged stereoselectivity in the enzyme reaction. HO exhibits a direct electronic effect in inducing the alpha-meso position for electrophilic attack. The heme pocket residues, reflecting the distal steric effect on the tilt of coordinated cyanide, differ for the alpha- and gamma-meso orientations.

An analysis of the resonance Raman (RR) spectra for ferrous yeast iso-1 cytochrome c and its meso-d4, pyrrole-15N,2,4-di(a-d1) and 2,4-di(b-d2) isotopomers, derived from enzymatic coupling of apoprotein with labeled hemes, involves the assignment of RR bands to every porphyrin in-plane skeletal mode and several out-of-plane skeletal modes. Isotope shifts of the 2, 4-thioether substituent modes provide assignments upon preferential deuteration. Some powerful bands in the B-resonant spectrum are the C-S stretching bands of these substituents.

A mixture of tetraarylporphyrins is formed by the self-condensation of 5-aryldipyrromethane with an aryl-substituted one-carbon bridging unit. This reaction is studied to determine MacDonald-type 2 + 2 condensation and develop four new synthetic pathways to meso-tetraarylporphyrins. Some of these products are useful in the synthesis of 5, 10, 15, 20-tetraaryl-substituted porphyrins with 2-field rotational symmetry.

Metal-catalyzed oxidative cyclizations of 1- and 19-substituent carrying a, c-biladiene salts results in the formation of unique macrocycles. The Cu(II) catalyzed cyclization of biladiene salts results in a macrocycle whose Cu(II) complex contains a 1-propionate valley group. Cr(III)-catalyzed oxidative cyclization of biladienes yield metal-free prophyrins have the reaction pathway similar to electrochemical route.

The aggregation properties of a series of 5-nitro derivatives of octaethylporphyrine were investigated. Results showed that strong pi-pi dimeric or linear aggregates of nitro-substituted metalloporphyrins were observed in the solid state. Analysis of several porphyrin molecules showed a requirement for an asymmetric electron distribution in the system. Weak aggregation was observed in the free-base porphyrin while intermediate to strong dimerization was shown by corresponding zinc complexes.

Extensive X-ray studies of six Ni(II) tetraphenylporphyrins (NiTPP) probe into the effects of beta-substituents on their conformation. The studies reveal nonplanar saddle conformations for four of the NiTPPs in solution, powder and crystal forms, planar shapes for two NiTPPs in powder form and, for one of the latter two, a planar shape in solution as well. The results suggest that porphyrin and hydroporhyrin structures are highly flexible in vitro and in vivo and that they may change their conformations in response to steric influences such as from beta-substituents.

The nickel and copper complexes of dodecasubstituted porphyrins bearing aryl groups at the meso positions and propano rings at the pyrrole beta positions were investigated. It was shown that the nickel complex NiTC5T(3,4,5-OMeP)P exhibits a nonplanar conformation, while the corresponding copper complex CuTC5T(3,4,5-OMeP)P is planar. The usefulness of spectroscopic probes of porphyrin nonplanarity is also investigated. The identification of the planar dodecasubstituted porphyrin indicates the flexibility of the tetrapyrrole macrocycle.

The magnetic circular dichroism (MCD) spectra of five- and six- coordinate chlorin complexes with nitrogenous axial ligands to ferric and ferrous ions are studied. The complexes examined are bis(1-methylimidazole)ferrous, (1,2-dimethylimidazole)ferrous, bis(1-methylimidazole)ferric and 1-methylimidazole/CO or NO compounds. The results show unique MCD signals for determining the presence of histidine or axial ligands in iron chlorine-containing protein.