Crystallization and preliminary X-ray crystallographic analysis of the putative [6Fe-6S] prismane protein from Desulfovibrio vulgaris (Hildenborough).

Crystals of the prismane protein from Desulfovibrio vulgaris (Hildenborough) containing a putative [6Fe-6S] cluster have been obtained and X-ray data collected to a resolution of 1.7 A using synchrotron radiation. The unit cell is orthorhombic with a = 64.1, b = 65.1 and c = 154.1 A, space group P2(1)2(1)2(1) (No. 19). The unit cell will readily accommodate four molecules of molecular weight 60 kDa with a corresponding solvent content of approximately 48%.

Reversible super-reduction of the cubane [4Fe-4S](3+;2+;1+) in the high-potential iron-sulfur protein under non-denaturing conditions. EPR spectroscopic and electrochemical studies.

The reversible 2 x 1 e- reduction of the cubane cluster from oxidized to reduced to super-reduced states ([4Fe-4S]3+<-->[4Fe-4S]2+<-->[4Fe-4S]1+) was studied in high-potential iron-sulfur proteins (HiPIPs). Super-reduction to the 1+ state was not observed in any of the seven HiPIPs tested during cyclic voltammetry (down to -0.95 V). However, equilibration at low potential (pH 7.5) of Rhodopila globiformis HiPIP yields a transient peak around -0.47 V due to the oxidation of super-reduced HiPIP adsorbed at the electrode. The peak area depends on the equilibration potential according to a one-electron Nernst curve with a half-wave potential at -0.91 V. Reduction of R. globiformis HiPIP with titanium (III)citrate at pH 9.5 is very slow [pseudo-first-order half-life of 23 min with a 100-fold excess Ti(III)] but is reversible, and the EPR spectrum with g values of 2.04 and 1.92 is similar to that of reduced [4Fe-4S]1+ ferredoxins. Chemical or electrochemical reoxidation of the super-reduced form resulted in an EPR spectrum with g parallel = 2.12 and g perpendicular = 2.03, i.e. identical to that of oxidized HiPIP. From the equilibrium concentration of super-reduced HiPIP at a low concentration of Ti(III), a reduction potential of -0.64 V can be estimated. Super-reduction of the large HiPIP (iso-2) from Rhodospirillum salinarum is also possible with Ti(III)(gz = 2.05) but the super-reduced state is unstable. No super-reduction with Ti(III) was observed for the other HiPIPs. The difference between the electrochemically observed reduction potential and oxidation potential is explained by a fast and reversible conformational change upon super-reduction. The rate of super-reduction with Ti(III) is limited by the small amount (0.1%) of HiPIP in the 2+ state with the super-reduced conformation.

Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine.

The Staphylococcus hyicus lipase is an acyl hydrolase with broad substrate specificity including neutral glycerides and phospholipids. To obtain further insight into the mechanism of action of this enzyme, we tested several sulfonyl fluorides as active site-directed inhibitors. The enzyme is resistant to the well-known serine protease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF), but is rapidly inactivated by hexadecylsulfonyl fluoride. The kinetics of inactivation were studied in Triton X-100 micelles. Inactivation is fast and the rate of inactivation is constant over the pH range where this lipase is active. Metal ions like Ca2+ and Sr2+ do not appreciably influence the rate of inactivation, although the enzymatic activity is significantly increased, suggesting a structural role for these ions. The S. hyicus lipase contains a consensus sequence G-H/Y-S-X-G. Substitution by site-directed mutagenesis of this serine (Ser369) by a cysteine resulted in a mutant with only 0.2% residual activity. The activity of this mutant could not be inhibited with water-soluble sulfhydryl reagents either in the presence or absence of Triton X-100 micelles. In the presence of Triton X-100 micelles, inactivation of the mutant occurred with 4-nitrophenylhexadecyl disulfide (t1/2 = 125 min) while the wild-type enzyme does not react at all. We conclude that Ser369 is the active site residue and that in water this residue is inaccessible. Only after interfacial activation Ser369 (or Cys369) becomes exposed and reacts with irreversible inhibitors.