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The thermodynamic parameters of the reduction of four different variants of Thiobacillus amicyanin by electrochemical techniques are discussed. They are determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion.

A molecular technique for studying the diversity of genes from complex microbial populations is presented. Bacteria obtained from Escherichia coli were analyzed by denaturing gradient gel electrophoresis. This approach separated polymerase chain reaction-amplified fragments of genes coding for 16S rRNA of the same length. The utility of this molecular approach for genetic research was established.

Laser flash photolysis method is used to study how small molecules like carbon monoxide and p-nitrophenol, which is a substrate-analogue inhibitor, affect the oxygen binding kinetics of the protein tyrosinase in Streptomyces antibioticus. The O2-binding rate constant and the initial intensity change increases with temperature and the larger intensity changes for higher temperatures may correspond to an enhancement in protein fluctuations, which increase the dissociation of the O2 molecule from the protein.

The prosthetic group of low-spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis-His and His–Met cytochromes to His–Lys co-ordination in the M100K mutant of Paracoccus versutus cytochrome c550. The electronic structure of the His–Lys haem is shown to be similar to that produced by His–cyanide co-ordination, such that NMR can be used to determine the geometry of the His ligand. [Copyright &y& Elsevier]

Discusses a study which examined the expression, purification and characterization of the soluble binuclear copper center domain of cytochrome c oxidase of Paracoccus versutus. Experimental procedures; Results and discussion.

The effects of two single-point cavity-forming mutations, F110S and I7S, on the internal dynamics of azurin from Pseudomonas aeruginosa were probed by the phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)) whereas more general effects on structural fluctuations were deduced from the phosphorescence acrylamide quenching rate constant (k(q)), which measures the diffusion of the solute through the protein fold. The results show a spectacular, 4-5 orders of magnitude, increase of k(q) emphasizing that large amplitude structural fluctuations permitting acrylamide migration to the protein core have been drastically enhanced in each azurin mutant. The large, 12-15 kcal/mol, decrease in the activation enthalpy associated to k(q) suggests that the rate enhancement is caused, rather than through a generalized increase of protein flexibility, by the elimination of an inner barrier to the diffusion process. According to tau(0) the chromophore environment is more fluid with I7S but strikingly more rigid with F110S, demonstrating that when internal cavities are formed local effects on the mobility at the mutation site are unpredictable. Both tau(0) and k(q) reveal a structure tightening role of bound Cd(2+) that correlates with the increase in stability from apo- to holo-azurin. While these alterations in internal dynamics of azurin do not seem to play a role on electron transfer through the central region, the enhanced migration of acrylamide emphasizes that cavities may be critical for the rapid diffusion of substrates to buried, solvent inaccessible sites of enzymes.

The prosthetic group of low-spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis-His and His-Met cytochromes to His-Lys co-ordination in the M100K mutant of Paracoccus versutus cytochrome c(550). The electronic structure of the His-Lys haem is shown to be similar to that produced by His-cyanide co-ordination, such that NMR can be used to determine the geometry of the His ligand.