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The route of O₂to and from the high-spin heme in heme-copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB⁺ on the pathway to and from the high-spin heme. The presence of CO on CuB⁺ suggests that O₂binding may be compromised in CO flow-flash experiments. Time-resolved optical absorption studies show that the rate of O₂and NO binding in the bovine enzyme (1 × 10⁸M⁻¹s⁻¹) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t½ = 1.5μs) of CO from CuB⁺. In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O₂and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 10⁹ to 1 × 10⁸M⁻¹s⁻¹), but is still considerably faster (~10μs at 1atm O₂) than the CO off-rate from CuB in the absence of O₂(milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O₂and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB⁺ impedes the binding of O₂to CuB⁺ or, if O₂does not bind to CuB⁺ prior to heme a3, whether the CuB⁺-CO complex sterically restricts access of O₂to the heme. Both possibilities are discussed, and we argue that O₂binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB⁺ in a concerted manner through steric and/or electronic effects. This would allow CuB⁺ to function as an electron donor during the fast (5μs) breaking of the OO bond. These results suggest that the binding of CO to CuB⁺ on the path to and from heme a3 may not be applicable to O₂and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

Research has been conducted on heme-copper oxidases. The covalently linked structure of this oxidase has been investigated via the use of infrared spectroscopy, synthetic model studies, normal mode calculations and isotopic substitution on tyrosine and histidine, and the results are reported.

Research has been conducted on the heme-copper respiratory oxidases. The redox-linked proton-pumping mechanism of these oxidases have been investigated via the use of cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared spectroscopy and the results are discussed.

Research has been conducted on the binuclear copper complex. The electronic structure of this complex has been investigated via the resonance Raman and electroabsorption spectroscopy and the results indicate that the crystallographic Cu (sub)2 distances determined for the nitrate and acetate salts are consistent with the metal-metal interaction.

Results demonstrate that water is the axial ligand of the deoxy heme iron in the H93G proximal cavity myoglobin mutant. Data show that the CO adduct of the protein is a six-coordinate with a histidine trans ligand and it functions as a conformationally driven ligand switch.

UV-visible absorption and magnetic circular dichroism (MCD) data were gathered for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 deg C. Findings showed that H93G myoglobin's heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0 and a mixture of species at pH 7.0- including five-coordinate hydroxide-bound, and six-coordinate structures.

Research was conducted to examine intertriad heterodimeric ((OEP)MoOs(OEP)), ((OEP)WRu(OEP), ((OEP), MoOs(TPP)))PF6 and ((OEP)WRu(TPP))PF6 metalloporphyrins using solution and X-ray crystallographic spectroscopic methods. The magnetic properties of the neutral species and the structural conformation of the corresponding monocations indicate a molecular orbital description. Results suggest that steric factors may have a significant role in the exact description of the complexes and that their metal-metal bond strengths are similar.

Research was conducted to examine various spectroscopic studies of WOs and MoRu porphyrin dimers using variable-temperature 1H NMR, resonance Raman, magnetic susceptibility and X-ray crystallography. The objective was to determine a proper molecular orbital diagram for the dimers. Results demonstrate an unusual molecular orbital diagram which has never been observed before in metal-metal bonded metalloporphyrin dimers.

Vibrational data on deoxyferrous and ferrous-CO derivatives of the proximal histidine ligands in Notomastus lobatus chloroperoxidase (NCPO) and Amphitrite ornata dehaloperoxidase (DHP) are similar to data reported for neutral histidine/imidazole-ligated globins and heme complexes. This indicates that the proximal histidine ligands of NCPO and DHP do not have a special catalytic role in the two enzymes' mechanisms of action. It is possible that DHP and NCPO rely on another distal mechanism to generate the oxoferryl intermediate.

The rotational barrier for MoW(meso-monotolyl octaethylporphyrin)2 (((TOEP)MoW(TOEP))) was studied through variable temperature nuclear magnetic resonance spectroscopy and complete band shape analysis. Findings suggested the presence of competing effects on the determination of heterometallic bond strengths. Atomic orbital energy mismatch caused by a difference in metal atom electronegativities resulted into a net loss of Mo d(sub xy)-W d(sub xy) interaction energy and therefore a delta-bond which is slightly weaker in MoW than in either Mo2 or W2.

Most experimental studies on protein folding dynamics have been limited to timescales of 1 ms or more. It is clear that many phenomena necessary for folding of proteins occur in a much shorter time than that. Formation of tertiary and secondary structures can take place on microsecond and nanosecond timescales, respectively. The fast events are required and dominate at times, but with only a few exceptions the study of these events experimentally has been possible only lately. New approaches can start and monitor the fast events with temporal resolution going down to picoseconds. Some important results for folding of some globular proteins and polypeptide models are now available.

Time-resolved infrared spectroscopy was utilized to investigate the mechanism governing the folding dynamics for the 'I' form of apomyoglobin (apoMb-I). Results have shown that the structure of apoMb-I was kinetically and thermodynamically heterogeneous comprising of AGH core. The stability of the core was determined by the presence of native interhelical contacts and solvated helical portions without interhelical contacts. The unfolding time was dependent on the stability of the helical segment.

A study was conducted on connectivity between CuB and glutamic acid 286 (Glu286) in cytochrome bo3 from Escherichia coli. The findings indicate an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286. Moreover, this connectivity, which may be due to a chain of bound water molecules, may be of interest in the context of the proton pump mechanism of the heme-copper oxidases.

The group-state of cluster complexes of [M6X8Y6]2- with binary and ternary metal-halide compounds was analyzed by resonance Raman spectroscopy. The polarized (TBA)2[Mo6Cl8Cl6] complex exhibits two symmetric Raman bands (A1g) with an Mo6 breathing mode. The Mo and W cluster complexes also exhibited similar metal-halide force constants and the ratio of symmetric metal-halide Raman shifts are equal to the square root of the inverse mass ratios of halogens.

The oligomeric Ru2(O2C(CH2)6CH3)4 and its Rhsub2super4+ analogue exhibit extended pi-systems in noncoordinating solvents. The axial ligands of dimetal tetracarboxylate systems are affected by dipolar and contact shifts. The presence of direct pi-delocalization in pi-type ligands indicates the formation of conductive polymers through bridging pi-type ligands. This link connects Ru2(o2CR)sub4supern+ units in dimetal tetracarboxylate systems. Downfield sigma-contact contributions occur due to the chemical shifts on axial THF ligands and equatorial carboxylate ligands.

A synthetic myoglobin was created to study the formation of six-coordinate complexes in the presence of nitric oxide. When nitric oxide binds with the heme protein, it typically weakens the nearest bond between heme and iron. In contrast, the synthetic myoglobin contains imidazole that functions as a ligand for iron. The presence of nitric oxide breaks this bond to form a five-coordinate complex. The six-coordinate complex can only be formed in the presence of excess imidazole, which shows that it has a lower binding constant than nitric oxide for the heme.

The dynamics of protein folding was observed in a small 21-residue alpha-helical alanine-based peptide. The rate of protein unfolding during helix melting in this peptide was generated using laser-induced temperature jump kinetics and probed using time-resolved infrared spectroscopy. The peptide exhibited fast unfolding kinetics within 20 nanoseconds with a time constant of 160 (+/-) 60 nanosecond at 28 degrees Celsius when treated with a laser-induced temperature jump of 18 degrees Celsius. The folding rate constant of this peptide was determined during helix formation at 28 degrees Celsius.

The water soluble hydrogenase of the bacteria Chromatium vinosum was studied spectroscopically and eight different states of the nickel ion in the enzyme were observed. All the enzyme states showed two weak bands and one strong band and the distance of the bands from each other was fixed. The position of these bands however changed according to the charge density of the nickel ion. It has been proposed that these bands are due to a non-protein group or groups which are present near the nickel ion and are sensitive to redox state of the nickel ion.

X-ray absorption spectrum of recombinant CuA-binding domain of Bacillus subtilis cytochrome oxidase indicates the existence of a direct Cu-Cu bond in the CuA center for cytochrome oxidase. Modified type 1 copper centers constitute the CuA sites in cytochrome oxidase and N2O reductase. The modified type 1 copper centers have one cysteine, one histidine and one ligand which is weakly bound.

Infrared studies of the interactions between carbon monoxide and nickel (II) in hydrogenase isolated from Chromatium vinosum reveals a 2060 cm(-1) band due to photolyzable CO-Ni binding and additional infrared lines that arise from non-CO bonds co-ordinated to the Ni active site. Infrared spectroscopy provides structure-specific information on redox and co-ordination environment of Ni site in EPR silent states of hydrogenase.

Thermus thermophilus bacteria yield cyano and unliganded derivatives of cytochrome ba3, and the process is analyzed by resonance Raman, ultraviolet and electroparamagnetic resonance spectroscopies. Cytochrome ba3 is an isolated, unliganded enzyme with structural similarities to the bovine cytochrome aa3.

Picosecond time-resolved spectroscopic studies on the reaction steps following photodissociation of carbon monoxide (CO) from cytochrome c oxidase's a3-CuB site reveal that CO transfers completely and the CuBCO complex forms within one picosecond. CO which is bound to a3 has an orientation angle of 20 degrees to the normal. The CO dipole has an orientation of about 55 degrees of the heme normal on binding with CuB.

An examination of the photochemistry of flash-induced photodissociation and the rejoining of carbon monoxide in cytochrome aa3-CO reveals that the secretion of ligand L from CuB may be caused by the picosecond association of photodissociated CO with CuB. During the attachment of various tiny molecules, especially O2 at the active region, 'ligand shuttle' may take place.

Ligand attaching activities of cytochrome bo, a heme-copper oxidase from Escherichia coli which behaves as an ubiquinol oxidase, were examined. Ligation interactions of heme-copper oxidase require protein structural characteristics and the CuB region where the first ligation process in the physiological activity of heme-copper oxidases occurs.

Electron transfer vibrational excitation and relaxation in the mixed-valence transition-metal dimer ((NC)5MIICNMIII(NH3)5)- (M = Ru, Os) were studied using picosecond infrared spectroscopy. Excited state redox isomer ((NC)5MIIICNMII(NH3)5)- is developed upon optical excitation into the metal-to-metal charge transfer bond. The ground state is developed again after back electron transfer. Massive quantities of energy are introduced into the terminal MC triple bonded to N stretching mode, upon the switching back to the ground electronic state.

The far-infrared spectra of complexes of the type Mo2X4L4, where X = Cl, Br or I, L = PMe3, PEt3, P(n-Pr)3 or P(n-Bu)3 or AsMe3 are determined to elucidate their vibronic, electronic and molecular structures. The assignment of the skeletal vibrational modes is hampered by the former's co-existence with internal deformation modes in the same spectral region and by the substantial mixing of modes of the same symmetry. Nevertheless, assignments for b2-symmetry modes are conclusive.

The reaction of cyanide with cytochrome ba(sub 3) of Thermus thermophilus was studied. Spectroscopic analysis showed that hydrogen cyanide partially reduces ba(sub 3), as well as coordinating to the cytochrome a(sub 3) center to form a paramagnetic complex. This complex has a composition of Fe(II)(sub a3)-CN.Cu(II)(sub B)-CN. The heme B copper of cytochrome ba(sub 3) is coordinated through four (super 14)N equatorial atoms, to provide a strong equatorial ligand field.