Structure function studies of the NDHD4 protein of the co₂ uptake mechanism in Synechococcus elongatus sp. pcc 7942

Abstract

To overcome the poor kinetic efficiency of Rubisco, cyanobacteria have evolved a CO2-concentrating mechanism (CCM) that maintains high carbonation levels in the cytoplasm. The cyanobacterial CCM utilizes a modified NADH dehydrogenase oxidoreductases (NDH-1⌄3,4) to hydrate CO2 to bicarbonate (HCO⌄3-) using photosynthetic redox energy. The energetic coupling effectively captures the inorganic carbon (C⌄i) in the cell in a form that is efficiently used within the carboxysome for fixation into sugars. The coupling mechanism by NDH-1⌄3,4, along with the rest of the CCM, acts as a supercharger for CO2, saturating Rubisco’s active sites and avoiding photorespiration.

Here, I test the hypothesis that proton-pumping by the antiporter-like NdhD4 subunit of the NDH-1⌄4 complex is essential for coupling of CO2 hydration to NDH-1 electron transfer. Structural modeling shows that amino acids involved in proton pumping in other species (e.g., E. coli) are conserved in NdhD4. The overall carbonic anhydrase (CA)-like reaction in the Zn-containing CupB subunit is somehow coupled to an unknown energy source to drive the reaction far-from-equilibrium in favor of bicarbonate production. By analogy with known carbonic anhydrases (CAs) and characteristics of CupB mutants, we hypothesize that the NdhD4 protein functions to trap and remove protons produced in the CO⌄2- hydration reaction by pumping them across to the luminal side of the membrane away from the anhydrase active site and avoiding the back-reaction and driving the reaction in favor of HCO⌄3- production. To probe the NdhD4 subunit’s role in CO2 hydration, I identified and mutated several residues that likely effect the proton trapping and translocating ability of the subunit. When mutated, these residues alter the photosynthetic ability of the cell, indicating that they are critical for optimal proton translocation and therefore CO2 hydrating.