Abstract
Oxygenic photosynthesis is the basis for aerobic life on earth. The catalytic Mn(4)O(x)CaY(Z) center of photosystem II (PSII), after fourfold oxidation, extracts four electrons from two water molecules to yield dioxygen. This reaction cascade has appeared as a single four-electron transfer that occurs in typically 1ms. Inevitable redox intermediates have so far escaped detection, probably because of very short lifetime. Previous attempts to stabilize intermediates by high O(2)-back pressure have revealed controversial results. Here we monitored by membrane-inlet mass spectrometry (MIMS) the production of from (18)O-labeled water against a high background of in a suspension of PSII-core complexes. We found neither an inhibition nor an altered pattern of O(2) production by up to 50-fold increased concentration of dissolved O(2). Lack of inhibition is in line with results from previous X-ray absorption and visible-fluorescence experiments, but contradictory to the interpretation of previous UV-absorption data. Because we used essentially identical experimental conditions in MIMS as had been used in the UV work, the contradiction was serious, and we found it was not to be resolved by assuming a significant slowdown of the O(2) release kinetics or a subsequent slow conformational relaxation. This calls for reevaluation of the less direct UV experiments. The direct detection of O(2) release by MIMS shows unequivocally that O(2) release in PSII is highly exothermic. Under the likely assumption that one H(+) is released in the S(4)→S(0) transition, the driving force at pH 6.5 and atmospheric O(2) pressure is at least 220meV, otherwise 160meV.
Highlights
AInstitutionen för Kemi, Kemiskt Biologiskt Centrum (KBC), Umeå Universitet, Linnaeus väg 6, S-90187 Umea, Sweden; bMax-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D-45470 Mulheim an der Ruhr, Germany; and cAbteilung Biophysik, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49069 Osnabruck, Germany
It appears that reaction 1 occurs significantly faster than the formation of S4 from S3YZox. For decades it has appeared as if the four electrons were transferred in reaction 1 in one batch, because three different events proceed with the same half-rise time, typically 1–1.5 ms, namely (i) the appearance of O2 in solution, (ii) the reduction of YZox, and (iii) the reduction of the Mn4OxCa cluster in the S3 state. This coincidence has been well documented by a wealth of independent techniques. (i) The appearance of O2 in solution was time resolved by four different techniques, namely continuous flow [9], EPR [10, 11], polarography with a bare Pt electrode [2, 12,13,14] and via absorption changes of intracellular cytochrome c oxidase [15]. (ii) The reduction of YZox was detected by timeresolved EPR [10, 16, 17], and (iii) the reduction of the Mn4OxCa cluster by UV absorption [7, 12, 18,19,20], delayed chlorophyll fluorescence (DF) [21], and time-resolved Mn-K-edge spectroscopy [8, 22]
The equilibration of photosystem II (PSII) suspensions in a buffer containing 100 mM sucrose, 25 mM CaCl2, 10 mM NaCl, 1 M glycine betaine, 50 mM MES and 30–50% H218O with O2 or N2 at the applied pressure in the gas phase of a specially developed reaction cell (Fig. 1) was monitored by membrane-inlet mass spectrometry (MIMS)
Summary
AInstitutionen för Kemi, Kemiskt Biologiskt Centrum (KBC), Umeå Universitet, Linnaeus väg 6, S-90187 Umea, Sweden; bMax-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D-45470 Mulheim an der Ruhr, Germany; and cAbteilung Biophysik, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49069 Osnabruck, Germany. The catalytic centre of O2 production in PSII, coined the OEC (oxygen evolving complex), comprises the manganese–oxygen–calcium (Mn4OxCa) complex and its ligands, which include two substrate “water” molecules of undefined protonation state It includes a redox-active tyrosine residue, coined tyrosine ZðYZÞ, which is the essential electron transfer link to the photoactive reaction center of PSII. For decades it has appeared as if the four electrons were transferred in reaction 1 in one batch, because three different events proceed with the same half-rise time, typically 1–1.5 ms, namely (i) the appearance of O2 in solution, (ii) the reduction of YZox, and (iii) the reduction of the Mn4OxCa cluster in the S3 state. A direct four-electron transfer from water to the Mn4OxCaYZox moiety is improbable, redox intermediates inevitably exist, and it is pivotal for understanding the mechanism of water oxidation to characterize their chemical nature
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