Virtually all of the carbon in the biosphere is the result of carbon dioxide (CO2) fixation by the photosynthetic enzyme, D-ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco also has the dubious honour of being the most abundant protein on earth, a consequence of its woeful catalytic efficiency. Rubisco is both very slow and poorly selective for its substrate CO2, binding also oxygen (O2) and catalysing a competing wasteful oxygenation reaction. As one would expect Rubisco to have been subjected to extreme evolutionary pressures, its incompetency is a great puzzle. Understanding the reasons offers huge potential as a basis for reengineering Rubisco; even modest improvements in efficiency have major implications for improving light, water and nutrient utilization by plants, and, hence, applications for better agricultural crops, greening deserts and degraded land, and for soaking up green-house gases.
As Rubisco catalyzes a multi-step reaction involving as many as four enzyme-bound intermediates, whose instabilities give rise to multiple side reactions which further compromise its efficiency, it is possible that the enzyme's complex structure and function is a compromise solution to effecting quite difficult chemistry. Not surprizingly, much of this chemistry is not approachable by experiment. This provides a (very challenging) opportunity for computer simulation to try to define intractable issues, particularly the states and roles of the "invisibles" - protons and water molecules. Thus: protonation states of the forest of ionizable residue sidechains in the active site at different stages of the reactions (both carboxylation and oxygenation), as well as the protonation states for the reactant, intermediates and transition states; identities of the proton donors for the various steps; networks for channelling protons produced in the reaction away from the reaction centre; and origin of the water molecule consumed and produced (oxygenation only). We have addressed these issues using a range of computational methods, particularly ab initio QM and ONIOM QM/MM studies of active-site fragment complexes, and MD simulations with hybrid QM/MM potentials, with a view to delineating the mechanism as a basis for re-engineering of Rubisco.
|