No abstract

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3–7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 μs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

Anaerobic CO dehydrogenases catalyze the reversible oxidation of CO to CO2 at a complex Ni-, Fe-, and S-containing metal center called cluster C. We report crystal structures of CO dehydrogenase II from Carboxydothermus hydrogenoformans in three different states. In a reduced state, exogenous CO2 supplied in solution is bound and reductively activated by cluster C. In the intermediate structure, CO2 acts as a bridging ligand between Ni and the asymmetrically coordinated Fe, where it completes the square-planar coordination of the Ni ion. It replaces a water/hydroxo ligand bound to the Fe ion in the other two states. The structures define the mechanism of CO oxidation and CO2 reduction at the Ni-Fe site of cluster C.

The homodimeric nickel-containing CO dehydrogenase from the anaerobic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO to CO2. A crystal structure of the reduced enzyme has been solved at 1.6 angstrom resolution. This structure represents the prototype for Ni-containing CO dehydrogenases from anaerobic bacteria and archaea. It contains five metal clusters of which clusters B, B', and a subunit-bridging, surface-exposed cluster D are cubane-type [4Fe-4S] clusters. The active-site clusters C and C' are novel, asymmetric [Ni-4Fe-5S] clusters. Their integral Ni ion, which is the likely site of CO oxidation, is coordinated by four sulfur ligands with square planar geometry.

The CO dehydrogenase of the eubacterium Oligotropha carboxidovorans is a 277-kDa Mo-and Cu-containing iron-sulfur flavoprotein. Here, the enzyme's active site in the oxidized or reduced state, after inactivation with potassium cyanide or with n-butylisocyanide bound to the active site, has been reinvestigated by multiple wavelength anomalous dispersion measurements at atomic resolution, electron spin resonance spectroscopy, and chemical analyses. We present evidence for a dinuclear heterometal [CuSMo(AO)OH] cluster in the active site of the oxidized or reduced enzyme, which is prone to cyanolysis. C arbon monoxide dehydrogenases (CODHs) of aerobic or anaerobic Bacteria and Archaea, which represent the essential catalyst in the global biogeochemical cycle of atmospheric carbon monoxide (CO), catalyze the oxidation of CO to carbon dioxide (CO 2 ) or the reverse reaction [CO ϩ H 2 O 7 CO 2 ϩ 2 H ϩ ϩ 2 e Ϫ ] (1, 2). The annual removal of CO from the lower atmosphere and earth by microorganisms has been estimated to be Ϸ1 ϫ 10 8 tons (3). Thus, an important role of CODHs is to remove CO from the environment, helping to maintain the toxic gas at subhazardous concentrations.The Ni-containing CODHs from the anaerobic hydrogenogenic bacteria Carboxydothermus hydrogenoformans (4, 5) or Rhodospirillum rubrum (6) and the Mo-containing CODHs from the aerobic carboxidotrophic bacteria Oligotropha carboxidovorans (7-9) or Hydrogenophaga pseudoflava (10) have been structurally characterized. The homodimeric CODHs of C. hydrogenoformans or R. rubrum contain five metal clusters, of which clusters B, BЈ and a subunit-bridging, surface-exposed cluster D are cubane-type [4Fe-4S] clusters (5, 6). The active-site clusters C and CЈ of the C. hydrogenoformans CODH are asymmetric [Ni-4Fe-5S] clusters identified in the enzyme reduced with dithionite. Their integral Ni ion, which is the likely site of CO oxidation, is coordinated by four sulfur ligands with square planar geometry (5). Interestingly, the corresponding cluster of the CODH from R. rubrum has been described as an Fe mononuclear site in combination with an [NiFe 3 S 4 ] cubane (6).CODH from O. carboxidovorans consists of a dimer of LMS heterotrimers (7). Each heterotrimer is composed of a 17.8-kDa iron-sulfur protein (S), which carries two types of [2Fe-2S] clusters, a 30.2-kDa flavoprotein (M), which contains a noncovalently bound FAD cofactor, and an 88.7-kDa molybdoprotein (L), which harbors the active site of the enzyme. In a previous paper (7), a CODH preparation with a specific activity of 6.6 units͞mg was analyzed at a resolution of 2.2 Å. The enzyme's active site was modeled to contain Mo with three oxygen ligands, the molybdopterin cytosine dinucleotide (MCD) cofactor, and an SeH-group bound to the S␥ atom of Cys-388. In the present paper, we have applied multiple wavelength anomalous dispersion methods at up to 1.09-Å resolution to crystals containing fully functional CODH (23.2 units͞mg). The SeH-group could not be confirmed, and a Cu atom was identified instead, at ...