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The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed. protein engineering | selenocysteine insertion sequence element | selenoenzyme | stop codon suppression | X-ray crystallography

Electron paramagnetic resonance (EPR) spectroscopy is used for studying the thermally induced spin transitions in a family of heterospin polymer chain complexes of [Cu.sup.2} hexafluoroacetylacetonate with two pyrazole-substituted nitronyl nitroxides. The studies have shown an efficient tool for characterizing the spin transitions and for understanding the temperature-dependent sign and value of the exchange interaction in strongly coupled spin triads.

The exchange coupling of spin triads is analyzed in the complexes of [Cu.sup.2+] hexafluoroacetylacetonate with two pyrazole-substituted nitronyl nitroxides. The modulation of exchange interaction between copper and nitroxides by lattice vibrations lead to the spin exchange process.

The first stable, axial ligand mutations of a Cu(sub A) center in the Thermus thermophilus fragment of cytochrome ba(sub 3) have been realized. The mutants, Met160Gln and Met160Glu, show changes in the S-Cu charge transfer doublet centered near 500 nm and the near infrared transition at 790 nm. Both also have greater intensities and blue shifts of the higher energy CT partner of the doublet relative to wt. The blue shift of the S-Cu CT band in Met121Gln is consistent with a sulfur-to-oxygen substitution and a stronger ligand field.

Research into the impact of axial ligand mutation on the Cu(sub A) site in the recombinant water soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase ba3 is presented. It is concluded that the axial ligand has a small but significant impact on the spin distribution in the Cu(sub A) centre, reflecting on the ground-state wave function.