Low-temperature kinetic measurements of microsecond freeze–hyperquench (MHQ) cytochrome oxidase monitored by UV–visible spectroscopy with a newly designed cuvette

A special cuvette was designed to measure optical changes of MHQ (microsecond freeze–hyperquench) powder samples [Wiertz, Richter, Cherepanov, MacMillan, Ludwig and de Vries (2004) FEBS Lett. 575, 127–130] at temperatures below approx. 250 K. Reduced cytochrome c oxidase from Paracoccus denitrificans was reacted with O₂ for 100 μs, frozen as a powder and transferred to the cuvette. Subsequently, cytochrome oxidase was allowed to react further following stepwise increments of the temperature from 100 K up to 250 K while recording spectra between 300 and 700 nm. The temperature was raised only when no further changes in the spectra could be detected. The experiment yielded spectra of the A, P_M, F and O intermediate states. This demonstrated that the catalytic cycle of cytochrome oxidase at low temperature is similar to that at room temperature and so verifies the suitability of this method for the study of enzymes with high catalytic-centre activity.