Evidence That Bicarbonate Is Not the Substrate in Photosynthetic Oxygen Evolution

Abstract

(J.C., W.J.); and Max-PlanckInstitut fü r Bioanorganische Chemie, 45470 Muelheim an der Ruhr, Germany (K.B., J.M.)It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO 2 is an intermediate product of oxygen production in addition to O 2 . In this work, we investigated this possible role of exchangeable HCO 3 2 in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn 4 Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO 2 . This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO 2 pressure over ambient conditions did not affect the UV transient, whereas the S 3 / S 4 / S 0 transition was half-inhibited by raising the O 2 pressure only 10-fold over ambient, as previously established. (2) The flash-induced O 2 and CO 2 production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H 2 18 O enrichment. Light flashes that revealed the known oscillatory O 2 release failed to produce any oscillatory CO 2 signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO 2 an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.

Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation

“…Despite numerous experiments this point remained unresolved (21,22,35,38). Although PSII membranes isolated from spinach also possess some carbonic anhydrase activity (6,8), there is presently no evidence for a carbonic anhydrase directly connected to higher-plant PSII, and there is also no need for a CCM function (discussed above). Our finding that under continuous illumination the activity of PSII is nevertheless dependent on the HCO − 3 concentration of the buffer therefore demonstrates that HCO − 3 directly affects the activity of PSII.…”

“…The possible roles of inorganic carbon, C i ðC i = CO 2 ; H 2 CO 3 ; HCO − 3 ; CO 2− 3 Þ, in this process have been a controversial issue ever since Otto Warburg and Günter Krippahl (5) reported in 1958 that oxygen evolution by PSII strictly depends on CO 2 and therefore has to be based on the photolysis of H 2 CO 3 ("Kohlensäure") and not of water. These first experiments were indirect and, as became apparent later, were wrongly interpreted (6)(7)(8). Several research groups followed up on these initial results and identified two possible sites of C i interaction within PSII (reviewed in refs.…”

Crystal Structure and Functional Characterization of Photosystem II-Associated Carbonic Anhydrase CAH3 in Chlamydomonas reinhardtii

“…This value is then compared to the uncatalyzed rate in the same assay solution. The first-order rate constants extracted from the time courses of the isotopic equilibration reactions are a highly sensitive approach for relative estimations of CA activities in different samples, as well as for comparison with the uncatalyzed (background) isotopic equilibrium (Tu et al, 1986;Clausen et al, 2005).…”

Focusing the view on nature's water-splitting catalyst

“…On the basis of EXAFS measurements on Br K substituted samples, it was recently suggested that Cl K is not a direct ligand of the Mn 4 cluster in the S 1 state (Haumann et al 2006). In contrast to Cl K , no sound evidence exists for HCO 3 K binding within the WOC, and it is also highly controversial what kind of role it may play during photosynthetic water oxidation (Klimov & Baranov 2001;Stemler 2002;Clausen et al 2005;Hillier et al 2006). EXAFS on solution samples does not allow extracting the orientations of the Mn-Mn and MnCa vectors.…”