Binding of ligands and spectral shifts in cytochrome c oxidase

Abstract

On addition of reductant (ascorbate plus NNN''N''-tetramethyl-p-phenylenediamine) to isolated cytochrome c oxidase (ox heart cytochrome aa3), in the presence of the inhibitors azide or cyanide, an initial partially reduced species is formed with absorption peaks at 415 nm, 445 nm and 605 nm, which slowly gives rise to the final half-reduced species in whose spectrum the 415 nm peak has disappeared and a new absorption is seen at 430-435 nm. In the absence of reductant, cyanide forms an initial complex with the enzyme with a spectrum similar to that of the uncombined form, which slowly changes into the low-spin cyanide form with a peak at 432 nm. Azide, in absence of reductant, shifts the Soret peak slightly, but the resulting complex, which is probably thermally mixed-spin, undergoes no further changes. The Soret-peak shift of oxidized cytochrome a3 which occurs on reduction of the enzyme in the presence of azide is accompanied by a concurrent blue shift of the ferrous cytochrome a peak from 605-603 nm. A partial blue shift of the .alpha.-peak occurs in the half-reduced sulfide-inhibited enzyme, and a complete blue shift is seen in the analogous complexes with alkyl sulfides [a2+a33+HSR compounds, where R = CH3, C2H5 or (CH3)2CH]. Analogous spectroscopic effects with the ligands imidazole and alkyl isocyanmides suggest that on reduction of cytochrome a an interaction occurs between the 2 heme groups involving a high- to low-spin change in cytochrome a3, and after this, a change in the molecular environment of the cytochrome a. The latter effect, possibly a decrease in the hydrophobicity of the heme pocket, requires that the ligands on cytochrome a3, have a bulky and partially hydrophobic character.