A proposal for the metal geometry in yeast superoxide dismutase based on results from EXAFS spectroscopy.

Extended x-ray absorption fine structure (EXAFS) spectra have been recorded at the Cu edge and Zn edge in native yeast superoxide dismutase and at the Cu edge and Cd edge in the yeast superoxide dismutase derivative, where Zn has been substituted with Cd. Two different metal ligand distances in the range 1.9-2.0 A and 2.3-2.4 are determined for the Cu and Zn metals. For Cd in the Zn site two different metal ligand distances about 2.2 A and 2.6 A, respectively, were found. The striking feature is the similarity between the amplitude and radii determined for both the Cu and Zn sites. The increased distances for Cd can be explained by the increased ionic radius of Cd relative to Cu and Zn. Based on these EXAFS results and other relevant knowledge about the metal geometries, we propose that histidine 61 (63) positioned between the Cu and Zn metals are in one subunit bound to Zn and in the other to Cu. This model explains the recently observed difference between the two metal sites in each subunit.

Activation of mammalian skeletal-muscle carbonic anhydrase III by arginine modification.

Purified carbonic anhydrase isozymes I, II, and III (CA I, CA II, CA III) from various sources were treated with 2,3-butanedione and their bicarbonate dehydration reactions followed. The specific activities of human and bovine CA I and CA II and chicken CA III were not affected by the butanedione treatment, whereas the activities of human, gorilla, and bovine CA III were rapidly activated. These findings suggest that one, or both, of the two arginyl residues which appear to be unique to the active sites of the mammalian CA III isozymes are modified by butanedione.

The exchange of histidine C-2 protons in superoxide dismutases. A novel method for assigning histidine-metal ligands in proteins.

The rates of exchange of the C-2 protons of histidine residues in copper-zinc superoxide dismutase are substantially decreased by metal ion binding. This observation was used to distinguish between ligand and non ligand histidine residues in bovine and yeast copper-zinc superoxide dismutases; the effect was shown to depend only on metal ion co-ordination and not as a consequence of concomitant changes in protein structure. Selective deuteration of the zinc-only proteins at pH (uncorrected pH-meter reading) 8.2 and 50 degrees C resulted in the distinction between copper and zinc ligand resonances in the 1H n.m.r. spectrum of the enzymes. This method is proposed as a generally applicable technique for identifying histidine residues as ligands in metalloproteins.

Low and high pH form of cadmium carbonic anhydrase determined by nuclear quadrupole interaction.

The pH dependence of the nuclear quadrupole interaction between the excited 247-keV state in 111Cd bound to the active site in human carbonic anhydrase B and the nearest protein surroundings has been studied by means of the nuclear spectroscopic technique of perturbed angular correlation of gamma rays. The enzyme has been studied in the pH region 5.6-11.0 at 22 and -196 degrees C. The results show that the Cd enzyme changes from one form at low pH to another form at high pH both at 22 and -196 degrees C. The pK of the transition is 8.9 +/- 0.2 at -196 degrees C and close to 9 at 22 degrees C. Parallel to this transformation, the esterase activity of the Cd enzyme for the hydration of p-nitrophenyl acetate exhibits a pH dependency with a pH of 9.1 +/- 0.2. The sulfonamide inhibitor acetazolamide completely inhibits this activity of the Cd enzyme. The quadrupole interaction parameters for the Cd enzyme are not significantly different at -196 degrees C from those obtained at 22 degrees C. A measurement at 0 degrees C pH 5.7 shows, however, a form different from those at 22 degrees C pH 5.6 and -196 degrees C pH 5.7. The change in the quadrupole interaction with pH is, in a simple model, consistent with an ionization of a metal-bound water molecule.

Circular dichroism-inhibitor titrations of arsanilazotyrosine-248 carboxypeptidase A.

Coupling of carboxypeptidase with diazotized arsanilic acid specifically modifies a single tyrosyl residue. Yet, owing to the fact that the resultant azoTyr-248 can form an intramolecular chelate with zinc, two different circular dichroism probes result: azoTyr-248 itself and the azoTyr-248-Zn chelate. Both are environmentally sensitive and, characteristically, each can signal the same or different perturbations, as is apparent from circular dichroic spectra. This dual probe function greatly magnifies the scope of these chromophores in mapping the topography of the active center with respect to sites of interaction of inhibitors (or substrates). Titration of the azoenzyme with a series of synthetic, competitive inhibitors, e.g., L-benzylsuccinate, L-phenyllactate, and L-Phe, and with the pseudosubstrate, Gly-L-Tyr, in turn generates characteristic circular dichroic spectra. Their analysis yields a single binding constant for each of these agents, one molecule of each binding to the active center. Mixed inhibitions, as seen with beta-phenylpropionate and phenylacetate, resolved previously into competitive and noncompetitive components, are characterized by different spectral effects. Two molecules of these agents bind to the enzyme, consistent with both thermodynamic and enzymatic studies. The interactions leading to competitive and noncompetitive inhibition, respectively, can be recognized and assigned, based on the manner in which the extrema at 340 and 420 nm, reflecting azoTyr-248, and the negative 510-nm circular dichroism band, typical of its chelate with zinc, are affected and on the pH dependence of spectral and kinetic data. Certai4 noncompetitive inhibitors and modifiers induce yet other spectral features. Each probe is very sensitive to changes in its particular active center environment, though both can be relatively insensitive to inhibitors interacting at a distance from the active center.

Hydrogen-exchange study of the conformational stability of human carbonic-anhydrase B and its metallocomplexes.

In the range of pH 4.6--8.8, 25 degrees C, the apoenzyme of carbonic anhydrase B shows no evidence of any gross conformational changes, as studied by the hydrogen-deuterium exchange method. At pH 4.6 the addition of Co(II), Cd(II) or Mn(II) to the apoenzyme results in a destabilization of the native protein conformation, but in the range of pH 5.5--8.8 these metal ions, and Zn(II), slightly increase the conformational stability of the protein, in so far as they reduce the probability phi of solvent exposure of the peptide groups. In comparison with other proteins studied, native carbonic anhydrase is characterized by a rather compact conformation; for half of the peptide groups the probability of solvent exposure is less than 10(-4), corresponding to changes in standard free energy larger than 5.5 kcal mol-1 (23 kJ mol-1) following the conformational transitions by which these groups are exposed to the solvent.

Environment and conformation dependent sensitivity of the arsanilazotyrosine-248 carboxypeptidase A chromophore.

Reaction of carboxypeptidase A crystals with diazotized arsanilic acid uniquely modifies Tyr-248 to form a monazo derivative, which-in solution-forms an intramolecular inner-sphere coordination complex in the active site zinc atom. tarsanilazocarboxypeptidase exhibits spectral properties that are closely similar to those of the model complex, tetrazolylazo-N-carbobenzoxytyrosine Zn2+, with a distinctive maximum at 510 nm. In addition, its circular dichroic spectrum reveals a negative extremum at this wavelength, also characteristic of this complex. Both spectra are exquisitely responsive to pth changes and serve to monitor formation and dissociation of the metal-azophenol complex. Two pKapp at 7.7 and 9.5 delineate the pH range over which the probe characteristics most effectively gauge conformational features of the active center of arsanilazcarboxypeptidase. Other environmental parameters, e.g., substrates and inhibitors, as well as crystallization of the enzyme also critically influence the formation and dissociation of the complex; the response of the probe suggests that they induce conformational movement of the azoTyr-248 residue away from the zinc atom. tthe now available chemical, functional, structural data bearing on the spatial relationships of Tyr-248 and Zn, both thought critical to catalysis, are evaluated, based on spectra of arsanilazo- and nitrocarboxypeptidase crystals and solutions as well as on detailed kinetic analyses of the native enzyme in both physical states and based on the X-ray structure analysis of the native enzyme and its Gly-L-Tyr complex. Collectively all of the data show that the conformation of carboxypeptidase in crystals differs from that in solution. Moreover, reexamination of the original X-ray maps reported in 1968 and thought to preclude a Tyr-248-Zn interaction now leads to the conclusion that in up to 25 per cent of the molecules in the crystals ttyr-248 interacts with the active site zinc atom (W.D. Lipscomb (1973), Proc. Nat. Acad. Sci U.S. 70, 3797). Thus, even in the crystals the enzyme exists in at least two different conformations. In one of these Tyr-248 is near while in the other it is far from the zinc atom. The spectral effects of Gly-L-Tyr and beta-phenylpropionate on solutions of arsanilazo- and of nitrocarboxypeptidase demonstrate that during the catalytic process Tyr-248 moves away from the zinc atom. This implies a mechanistic role for Tyr-248 different from that postulated on the basis of X-ray crystallographic analysis. Indeed, the proximity of ttyr-248 to the zinc atom, when altered by substrates and inhibitor, may reflect certain of the properties characteristic of the entatic, active site.

Conformations of arsanilazotyrosine-248 carboxypeptidase A alpha, beta, gamma, comparison of crystals and solution.

The spectra of the alpha, beta, and gamma forms of zinc monoarsanilazotyrosine-248 carboxypeptidase A are indistinguishable. At pH 8.2 their crystals are yellow, while their solutions are red, lambda(max) 510 nm. Absorption and circular dichroism-pH titrations of the modified zinc and apoenzymes demonstrate that the absorption band at 510 nm is due to a complex between arsanilazotyrosine-248 and the active-site zinc atom. Two pK(app) values, 7.7 and 9.5, characterize the formation and dissociation of this arsanilazotyrosine-248.Zn complex. On titrations of the apoenzyme, the absorption band at 510 nm is completely absent at all pH values. Instead, there is a single pK(app), 9.4, due to the ionization of the azophenol, lambda(max) 485 nm. Substitution of other metals for zinc results in analogous intramolecular coordination complexes with absorption maxima and circular dichroism extrema characteristic of the particular metal. Similar data and conclusions have been derived from studies of heterocyclic azophenol.metal complexes. The present studies demonstrate that the conformation of the crystals of all generally available alpha, beta, and gamma forms of the arsanilazoenzyme differs from that of their solutions. The spectra of the modified x-ray crystals, however, differ from those of all other carboxypeptidase forms and crystal habits studied. The internal consistency of their data, their interpretation, and the conclusions of Lipscomb and coworkers [Proc. Nat. Acad. Sci. USA (1972) 69, 2850-2854] are examined. Dissimilar chemical modification or conformation is thought to underlie these differences. The arsanilazotyrosine-248.zinc complex is a sensitive, dynamic probe of environmental conditions. Its response to changes in pH and physical state of the enzyme suggest different orientation of the arsanilazotyrosine-248 side chain in solution from that in the crystal. This finding calls for reexamination of the basis of the substrate-induced conformation change which has been thought to be critical to the mechanism, postulated on the basis of the x-ray structure analysis performed at pH 7.5.

Differences between the conformation of arsanilazotyrosine 248 of carboxypeptidase A in the crystalline state and in solution.

Coupling of carboxypeptidase A crystals with diazotized arsanilic acid specifically labels tyrosine 248, an active-site residue of the enzyme. Many azophenols are yellow and their zinc complexes are red; the "yellow" absorption spectrum of zinc arsanilazocarboxypeptidase crystals is characteristic of the arsanilazotyrosyl group, not of the zinc complex. This is consistent with the interpretation of x-ray data on native crystals of carboxypeptidase A, indicating that tyrosine 248 and the zinc atom are too far apart to form a complex. However, the enzyme in solution is red, denoting the formation of a complex between zinc and arsanilazotyrosine 248. The most likely interpretation of the data is that the orientation of arsanilazotyrosine 248 in solution and in the crystal is different. If the unlabeled tyrosine 248 of native carboxypeptidase undergoes similar changes, these data may bear upon the low activity of the enzyme in the crystalline state and on the catalytic mechanism of the enzyme based on the crystal structure. The opportunities for analogous spectrochemical studies of other, similar systems are pointed out.