zene with Tl2+, Ag2+, SO4•-, and OH, respectively, were investigated in aqueous solution using optical and conductometric pulse radiolysis and in situ radiolysis ESR for detection. Tl2+ and Ag2+ were produced by reaction of OH with Tl+ and Ag+, respectively. SO4•was generated by reaction of eaq" with persulfate. Tl2+, Ag2+, and SO4•-react with the methoxylated benzenes by electron transfer to yield radical cations and Tl+, Ag+, and SO42-, respectively. In the absence of cyclohexadienyl radicals the radical cations decay by second-order kinetics with rate constants ranging from <4 X 103 to 1 X 109 M-1 sec-1 depending on the positions of the methoxy groups relative to each other. OH radicals react with the methoxylated benzenes by addition to the aromatic ring with diffusion-controlled rates. Depending on conditions, the hydroxycyclohexadienyl radicals thus formed subsequently undergo three different types of reaction: (a) bimolecular decay (k values ranging from 8 X 108 to 2 X 109 M-1 sec-1); (b) reaction with protons to yield radical cations (k values from 2 X 108 to 1.4 X 109 M-1 sec-1); and (c) reaction with radical cations (k values from 8.6 X 108 to 3.5 X 109 M-1 sec-1). The assignment of the optical absorption spectra of the radical cations and the determination of the respective extinction coefficients is based on a combination of optical and conductivity data. The radical cation yield obtained at pH 1 by reaction of protons with the OH adducts of the substrates amounts to >90% in the case of anisóle, 1,3-dimethoxybenzene, and 1,3,5-trimethoxybenzene.

The sodC-encoded Mycobacterium tuberculosis superoxide dismutase (SOD) shows high sequence homology to other members of the copper/zinc-containing SOD family. Its three-dimensional structure is reported here, solved by x-ray crystallography at 1.63-Å resolution. Metal analyses of the recombinant protein indicate that the native form of the enzyme lacks the zinc ion, which has a very important structural and functional role in all other known enzymes of this class. The absence of zinc within the active site is due to significant rearrangements in the zinc subloop, including deletion or mutation of the metal ligands His 115 and His 123 . Nonetheless, the enzyme has a catalytic rate close to the diffusion limit; and unlike all other copper/zinc-containing SODs devoid of zinc, the geometry of the copper site is pH-independent. The protein shows a novel dimer interface characterized by a long and rigid loop, which confers structural stability to the enzyme. As the survival of bacterial pathogens within their host critically depends on their ability to recruit zinc in highly competitive environments, we propose that the observed structural rearrangements are required to build up a zinc-independent but fully active and stable copper-containing SOD.

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The CuZn superoxide dismutases (SODs) from ox, sheep, pig and yeast were investigated by pulse radiolysis in order to evaluate the role of electrostatic interactions between O2.- and SOD proteins in the mechanism of action of the SOD enzymes. The protein net charge in this series varies, as evaluated by the protein pI values spanning over a large range of pH: 8.0 (sheep), 6.5 (pig), 5.2 (ox) and 4.6 (yeast). The amino acid sequences are largely conserved, with the three mammalian proteins being highly homologous and the yeast protein having some distinct variations in the region surrounding the active site. At pH 8.0 the activities of the SODs from various sources are similar, though the minor differences observed suggest that in the highly homologous mammalian series the most acidic protein is the most enzymically efficient one. The pH-dependences of the various activities in the pH range 7-12 are similar, and the related curves are best fitted by two pK values, which are approx. 9.2 and 11.0 for the mammalian enzymes and 9.1 and 11.4 for the yeast enzyme. The activities of the proteins at I 0.1 are decreased by approx. 20% when compared with the activity at I 0.02 at pH 8.5, whereas at pH above 10 the pH-dependence of the activity approaches that determined at I 0.02 and at pH 11.9 the activity is essentially independent of ionic strength. The dependence upon ionic strength also depends on the salt used, with perchlorate being more effective than phosphate or borate or Mops and still effective at pH above 10.5, where the effect of other salts becomes negligible. The dual and concerted dependence of the activities of different SODs on pH and salt concentration is explained with the encounter of O2.- with the active-site copper being governed by the protonation of two positively charged groups in the vicinity of the active site. The gradient between these localized charges and the rest of the protein may explain the different activities of the mammalian proteins at lower pH. On the basis of the sequence variation of the SODs examined it is not possible to definitely identify these groups. Likely candidates are conserved basic amino acid side chains in the vicinity (less than or equal to 1.2 nm) of the active site, i.e. Lys-134 and Arg-141, but co-ordination of OH- in the first copper co-ordination sphere may be an additional factor accounting for the higher pK.(ABSTRACT TRUNCATED AT 400 WORDS)