Rubisco activity is light regulated. Its activity declines rapidly to zero when the light is turned off and is regained only slowly when the light is once again turned on. Light activation is apparently indirect and involves complex interactions between Mg2+ fluxes across the thylakoid, CO2 activation, chloroplast pH changes, and an activating protein.
As noted in the previous chapter, light-driven electron transport leads to a net movement of protons into the lumen of the thylakoids. The movement of protons across the thylakoid membrane generates a proton gradient equivalent to 2.5 to 3.5 pH units and an increase in the pH of the stroma from around pH 7 to near pH 8.0. in vitro, rubisco is generally more active at pH 7. The Mg2+ requirement for rubisco activity was noted some years ago. Light also brings about an increase in the free Mg2+ of the stroma as it moves out of the lumen to compensate for the proton flux in the opposite direction.
Work in the laboratory of G. H. Lorimer, again using isolated Rubisco in vitro, has shown that rubisco uses CO2 not only as a substrate but also as an activator. The activating CO2 must bind to an activating site that is separate and distinct from the substrate-binding site. Based on these in vitro studies, Lorimer and Miziorko have proposed a model for in vivo activation that takes into account all three factors: CO2, Mg2+, and pH (Lorimer and Miziorko, 1980). According to this model, the CO2 first reacts with and ε-amino group of a lysine residue, forming what is known as a carbamate. Carbamate formation requires the release of two protons and, consequently, would be favored by increasing pH. The Mg2+ then becomes coordinated to the carbamate to form a carbamate-Mg2+ complex, which is the active form of the enzyme.
Further experiments, however, indicated that the in vitro model could not fully account for the activation of rubisco in leaves (Portis, 1990). In particular, measured values for in vivo mg2+ and CO2 concentrations and pH differences were not sufficient to account for more than half the expected activation level. This paradox was resolved by the discovery of an Arabidopsis mutant that failed to activate rubisco in the light, even though the enzyme isolated from the mutant was apparently identical to that isolated from the wildtype. Electrophoretic analysis revealed that the rca mutant, as it was called, was missing a soluble chloroplast protein. Subsequent experiments demonstrated that full activation of rubisco could be restored in vitro simply by adding the missing protein to a reaction mixture containing rubisco, RuBP, and physiological levels of CO2. This protein has moting light-dependent activation of rubisco.
The details of rubisco activase and how it operates are still being worked out, but it is known to require energy in the form of ATP. The protein has been indentified in at least 10 genera of higher plants as well as the green alga Chlamydomonas. It is clear that rubisco activase has a significant and probably ubiquitous role to play in regulating eukaryotic photosynthesis.
