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 ...

In eukaryotes, many essential secreted proteins and peptide hormones are excised from larger precursors by members of a class of calcium-dependent endoproteinases, the prohormone-proprotein convertases (PCs). Furin, the best-characterized member of the mammalian PC family, has essential functions in embryogenesis and homeostasis but is also implicated in various pathologies such as tumor metastasis, neurodegeneration and various bacterial and viral diseases caused by such pathogens as anthrax and pathogenic Ebola virus strains. Furin cleaves protein precursors with narrow specificity following basic Arg-Xaa-Lys/Arg-Arg-like motifs. The 2.6 A crystal structure of the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk)-inhibited mouse furin ectodomain, the first PC structure, reveals an eight-stranded jelly-roll P domain associated with the catalytic domain. Contoured surface loops shape the active site by cleft, thus explaining furin's stringent requirement for arginine at P1 and P4, and lysine at P2 sites by highly charge-complementary pockets. The structure also explains furin's preference for basic residues at P3, P5 and P6 sites. This structure will aid in the rational design of antiviral and antibacterial drugs.

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenylpyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an a-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.