Examination of 'high-energy' metastable state of the oxidized (OH) bovine cytochrome c oxidase: Proton uptake and reaction with H2O2.

Reoxidized cytochrome c oxidase appears to be in a 'high-energy' metastable state (OH) in which part of the energy released in the redox reactions is stored. The OH is supposed to relax to the resting 'as purified' oxidized state (O) in a time exceeding 200 ms. The catalytic heme a3-CuB center of these two forms should differ in a protonation and ligation state and the transition of OH-to-O is suggested to be associated with a proton transfer into this center. Employing a stopped-flow and UV-Vis absorption spectroscopy we investigated a proton uptake during the predicted relaxation of OH. It is shown, using a pH indicator phenol red, that from the time when the oxidation of the fully reduced CcO is completed (∼25 ms) up to ∼10 min, there is no uptake of a proton from the external medium (pH 7.8). Moreover, interactions of the assumed OH, generated 100 ms after oxidation of the fully reduced CcO, and the O with H2O2 (1 mM), result in the formation of two ferryl intermediates of the catalytic center, P and F, with very similar kinetics and the amounts of the formed ferryl states in both cases. These results implicate that the relaxation time of the catalytic center during the OH-to-O transition is either shorter than 100 ms or there is no difference in the structure of heme a3-CuB center of these two forms.

[Study of the interaction of DNA with copper ions using IR- and Raman spectroscopy]. / Izuchenie metodami IK- i Ramanovskoi spektroskopii vzaimodeistviia DNK s ionami medi.

DNA with Cu2+ ions in solution and films is studied by IR and Raman spectroscopy at different relative humidities (R.H. = 51 divided by 98%). DNA complexed with Cu2+ ions is shown to transit to the double-helix conformation, passing A-form, the water content per nucleotide (n) being essentially larger: 12 for Cu2+/P = 0.4 (n = 8 for DNA without ions). Cu2+ decrease the biopolymer hydration at R.H. 50-76% interactions with Cu2+ are stated to depend on the hydration degree of macromolecules. The analysis of the absorption band shifts of Raman spectra evidences the changes in the base torsion angle around the glycoside bond to the values characteristic of the syn-conformation and the interaction both with phosphates and nitrogen bases.

Conformational transitions of poly(dI-dC) in aqueous solution as studied by ultraviolet resonance Raman spectroscopy.

Poly(dI-dC) in aqueous solution can undergo different equilibrium geometries, which strongly depend on salt nature and concentrations. These equilibrium structures have been monitored by resonance Raman spectroscopy (RRS) measurements in the ultraviolet region, i.e. by using 257 and 281 nm laser excitation wavelengths which favor the resonance enhancement of the Raman contributions from inosine and cytosine residues of poly(dI-dC), respectively. Spectral changes depending on the NaCl concentration and on the presence of Ni2+ ions have been observed and interpreted in comparison with RRS results previously obtained for other alternating purine-pyrimidine polydeoxyribonucleotides, i.e. poly(dG-dC), poly(dA-dT) and poly(dA-dC).poly(dG-dT), which also showed B to Z conformational transitions in varying the salt concentrations. It is shown here that: i) the base stacking geometries are nearly the same in the high-salt form (5 M NaCl) of poly(dI-dC) as in the low-salt form (0.1 M NaCl) of the polymer, ii) however, the high-salt structure yields important differences from a B-helix (obtained in low-salt solution) as regards the nucleoside conformations (sugar puckering and base-sugar orientation), and: iii) the addition of 9 mM NiCl2 in the high-salt (5 M NaCl) solution of poly(dI-dC) induces the Z-conformation of the polymer.

Absorption and Raman spectroscopy study of cyt c-thiol complexes in acidic solutions.

Absorption UV-VIS and pre-resonance Raman spectra of acidic cyt c solutions with a series of thiols added (thiophenol, n-propanethiol, isopropanethiol, L-cysteine, dithiothreitol, 2-mercaptoethanol, N-acetyl-L-cysteine, p-acetamidothiophenol, 2-mercaptoethanamine, thioglycolic acid and mercaptopropionic acid), are presented. Interactions of cyt c molecule with the thiols were studied with the aim to identify binding of the thiols with the cyt c heme as its iron axial ligands. Absorption and Raman spectra showed some correlation between maxima of 700 nm region absorption band (typical for Fe-S axial bond in cyt c heme) and also wave numbers of spin state marker and axial ligand sensitive Raman bands on one, and pKa constant values of appropriate thiols on the other hand. These results imply thiol replacement of Met-80 from axial bond with heme iron and suggest that the force of Fe-L-cysteine axial bond is very close to the native axial bond (Fe-Met) for cyt c in neutral solution.