A short-lived intermediate in the photoenzymatic reduction of protochlorophyll(ide) into chlorophyll(ide) at a physiological temperature

The reduction of protochlorophyll(ide) into chlorophyll(ide) has been studied by flash absorption spectroscopy at 21°C, with a time resolution of 0.5 µs. The absorption changes have been recorded in the range 670–720 nm after the first and subsequent flashes given to an extract of etiolated bean leaves.

At 695 nm the flash-induced absorption increase has its maximum value immediately after the flash and then partly decays with a half-time of about 7–10 µs. A complementary behaviour is observed at 675 nm where the absorption change is very small at time zero and then increases to a stationary value with a half-time of about 6–9 µs. From measurements at several wavelengths it is concluded that a species with an absorption peak around 695 nm is formed immediately after the flash and is then transformed into a stable species with an absorption peak around 675 nm. Measurements at lower temperatures, down to—50°C, show that the transformation is much slowed down at decreased temperatures. The species absorbing at 695 nm (P695) is attributed to an intermediate in the photoreduction of protochlorophyll(ide) P639,650 into chlorophyll(ide) P676.

When the protochlorophyll(ide) is photoreduced before the flash illumination, the newly formed chlorophyll(ide) gets to a triplet state, which decays with a half-time of 15 µs at 21°C. This result shows that carotenoid molecules do not exert their protective role at this stage of chlorophyll (Chl) formation