Ligation and electronation states of cytochrome-c oxidase in relation to other oxidases and peroxidases.

ligation states Preparations of cytochrome-c oxidase (EC 1.9.3.1) isolated from bovine heart are usually heterogeneous. They can be considered as mixtures of two forms of the enzyme, ‘slow’ and ‘fast’, which differ considerably in their properties. Until recently preparations consisted mainly of ‘slow’ (‘resting’) oxidase. In this form haem a3 is entirely high spin, reacts slowly with cyanide and is reduced slowly by dithionite. In addition, ‘slow’ oxidase shows a unique e.p.r. signal arising from the binuclear centre, the ‘g’ = 12‘ signal. Baker et a]. [ 13 have shown that the appearance of the ‘slow’ form is prevented by maintaining a high pH (7.6-8.0) during the preparation of oxidase. ‘Fast’ oxidase as prepared can then be converted to ‘slow’ oxidase by incubation at low pH [ 1, 21. ‘Fast’ oxidase lacks the ‘g’= 12’ e p r . signal and has a Soret absorption maximum that is red-shifted relative to that of ‘slow’ oxidase. This latter observation reflects the presence of a thermal mixture of the high spin and low spin forms of haem a,. Haems a and a, in ‘fast’ oxidase are reduced essentially simultaneously by dithionite. When turnover is started by the addition of reductant (e.g. ascorbate/NNN’N’-tetramethyl-pphenylenediamine) to ‘slow’ oxidase under aerobic conditions Cu, and haem a are initially fullyreduced. There is then a gradual conversion to the ‘fast’ form, with a time-course similar to that observed for the reduction of haem a3 by the same reductant but under anaerobic conditions [ 31. The ‘slow’ to ‘fast’ conversion can be monitored as (a ) an increase in AA,,,-,,, ,,”,, which reflects a change in haem a3 from high spin to low spin [4]; and (6) a decrease in AAhOs-hZ3 ””,, which reflects a decrease in the steady-state level of haem a‘+ [3]. The copper-containing laccases, isolated from the lacquer tree (Rhus vernicifera) and a fungus (Polypoms verskolor), show remarkably similar behaviour. Like cytochrome oxidase, these enzymes catalyse the four electron reduction of dioxygen to water using electrons donated by a variety of substrates [5, 61. When turnover is started by addition