Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen

The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1) and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standard in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 2.1 : 1.8 : 0.005 : 0. In anaerobic cells, the ratios changed to 1.6 : 1.8 : 0.026 : 0.04 (PC : FNR : HYDA1 : Fdx5). Employing sodium dithionite and methyl viologen as electron donors the specific activity of hydrogenase in whole cells was calculated to be 382 ±96.5 μmolH2/min/mg. Importantly, these data demonstrate for competition of electrons from FDX1, HYDA1 is about 70-fold less abundant than its competitor FNR. Effective in vivo hydrogen production points to a channeling mechanism for FDX to HYDA1 electron transfer.