Studies of the Co 2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase by the use of spectrophotometry

Abstract

Transient optical absorption bands are formed upon addition of ribulose-1,5-bisphosphate to the Co 2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach and parsley. In the visible region, the prominent absorption band during steady state has a maximum at 610 nm. Stopped-flow technique was used to study the increase in absorbance at this wavelength, and two distinct phases in the progress curve for the approach to steady-state absorbance were observed. The rates for these two phases, respectively, were similar to those found earlier for the two enzyme-Co 2+-bound intermediates using EPR technique (Brändén et al. (1987) Biochim. Biophys. Acta 916, 298–303). It is therefore proposed that most of the transient optical absorption originates from an enzyme-Co 2+-coordinated ribulose-1,5-bisphosphate molecule and enzyme-Co 2+-coordinated enediolate anion of it, where bound ribulose-1,5-bisphosphate appears first. Furthermore, the most rapid phase in the progress curve is first-order reaction, independent of the ribulose-1,5-bisphosphate concentration. This indicates that the formation of enzyme-Co 2+-coordinated ribulose-1,5-bisphosphate is preceeded by another reaction in which ribulose-1,5-bisphosphate binds to the enzyme, probably without metal coordination.