Effect of calcium ions on electron transfer between hemes <Emphasis Type="Italic">a</Emphasis> and <Emphasis Type="Italic">a</Emphasis><Subscript>3</Subscript> in cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH₃) ₆ ³⁺ , three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a ₃ reduction. The difference spectrum of heme a ₃ reduction in the visible region is characterized by a maximum at ～612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a ₃ reduction is slowed down by low concentrations of Ca²⁺. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca²⁺ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca²⁺ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.     

Peroxidase activity of mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

Mitochondrial cytochrome c oxidase is able to oxidize various aromatic compounds like o-dianisidine, benzidine and its derivatives (diaminobenzidine, etc.), p-phenylenediamine, as well as amidopyrine, melatonin, and some other pharmacologically and physiologically active substances via the peroxidase, but not the oxidase mechanism. Although specific peroxidase activity of cytochrome c oxidase is low compared with classical peroxidases, its activity may be of physiological or pathophysiological significance due to the presence of rather high concentrations of this enzyme in all tissues, as well as specific localization of the enzyme in the mitochondrial membrane favoring accumulation of hydrophobic aromatic substances.     

Na(+)-induced reversal of the Ca2(+)-dependent red-shift of cytochrome a. Is there a hydronium output well in cytochrome c oxidase?

The Ca2(+)-induced red shift of the cytochrome a absorption spectrum is counteracted specifically by Na+ ions, whereas neither K+ nor Li+ do show comparable effect. At the same time Na+ does not reverse the H(+)-induced red shift of cytochrome a 2+. It is suggested that Na+ competes with Ca2+ for binding site(s) within the cytochrome oxidase output proton well communicating the heme a propionate substituent responsible for the Ca2(+)- or H(+)-induced red-shift of cytochrome a (Saari et al. 1980, J. Bioenerget. Biomembr. 12, 325-338) with the c-aqueous phase. The unusual ionic specificity of the well (Ca2+, Na+, proton) may point to H3O+ rather than H+ being the ion involved in proton conduction through the output well of cytochrome oxidase.     

A slow spectral shift of cytochrome c oxidase induced by EDTA and o-phenanthroline

Low concentrations of EDTA (in the presence of Ca2+ excess) or o-phenanthroline cause a blue shift of the oxidized cytochrome oxidase Soret absorption band. The effect develops within approximately 2 hours and does not depend on EDTA concentration provided the complexon is in a molar excess over the enzyme. It is suggested that the enzyme spectral characteristics depend on the presence of some tightly bound heavy metal ions which can stabilize one of the spectrally distinct conformations of cytochrome c oxidase.