The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.
Fluorescence quenching rate constants of some aromatic hydrocarbons by Zn2+, Ag", Cd2", In3+, SnZ+, Cs' , HgZ+, T1+, and PbZ+ were determined in aqueous and N,N-dimethylformamide solutions. Paramagnetic interactions, the heavy atom effect, and electron transfer were excluded as a possible quenching mechanism. Nz gas laser photolysis studies revealed that a cation radical of the fluorescer was detected only for N-ethylcarbazole quenched by Ag", PbZ+, and Hg2+ in N,N-dimethylformamide and for 1-pyrenesulfonic acid by Hg2+ in water. All other systems yielded the triplet state of the fluorescer quantitatively. The intermediates observed in the microsecond time region are the transient species with the lowest free energy. Picosecond laser photolysis of the Ag+ and PbZ+ quencher systems in N,N-dimethylformamide confirmed directly that the triplet state is induced by fluorescence quenching. On the basis of these results, it has been concluded that fluorescence quenching is due to nonfluorescent complex formation followed by rapid intersystem crossing. The electronic and geometrical structures of this complex were considered and compared to the excited aromatic hydrocarbon-halogen anion systems.
The crystal structure of Serratia protease from Serratia sp. E-15 was solved by the single isomorphous replacement method supplemented with anomalous scattering effects from both the Zn atom in the native crystal and the Sm atom in the derivative crystal, and refined at 2.0 A resolution to a crystallographic R-factor of 0.194. The enzyme consists of N-terminal catalytic and C-terminal beta-sandwich domains, as observed in alkaline protease from Pseudomonas aeruginosa IFO3080. The catalytic domain with a five-stranded antiparallel beta-sheet and five alpha-helices shares a basically common folding topology with those of other zinc metalloendoproteases. The catalytic zinc ion at the bottom of the active site cleft is ligated by His176, His180, His186, Tyr216, and a water molecule in a distorted trigonalbipyramidal manner. The C-terminal domain is a beta-strand-rich domain containing eighteen beta-strands and a short alpha-helix, and has seven Ca2+ ions bound to calcium binding loops. An unusual beta-sheet coil motif is observed in this domain, where the beta-strands and calcium binding loops are alternately incorporated into an elliptical right-handed spiral so as to form a two-layer untwisted beta-sandwich structure. The Ca2+ ions in the C-terminal domain seem to be very important for the folding and stability of the beta-sheet coil structure.
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