281 Acta Cryst. (1992). B48, 281-285 The Sensitivity of the Synchrotron Laue Method to Small Structural Changes: Binding Studies of Human Carbonic Anhydrase II (HCAII) BY M. LXNDAHL AND A. LILJAS Department of Molecular Biophysics, Chemical Center, University of Lund, Box 124, S 221 00 Lund, Sweden J. HABASH AND S. HARROP Department of Chemistry, University of Manchester, Manchester M13 9PL, England AND J. R. HELLIWELL Department of Chemistry, University of Manchester, Manchester M13 9PL, England, and SERC Daresbury Laboratory, Daresbury, Warrington WA4 4AD, England (Received 16 January 1991; accepted 18 November 1991) Abstract Human carbonic anhydrase II has been studied using Laue crystallography and short exposure times (3-20 s) at the Daresbury Synchrotron Radiation Source (SRS). Two types of crystals were investi- gated, the enzyme in complex with the inhibitor bisulfite and the enzyme at pH 6.0. A bisulfite ion and a water molecule, respectively, are bound with tetrahedral coordination to the zinc ion. These results have been subsequently confirmed by mono- chromatic methods. Synchrotron Laue crystallogra- phy is evidently able to characterize minor structural differences. Introduction The zinc enzyme carbonic anhydrase is widely spread among living organisms and at least seven genetically distinct isoenzymes are known in mammals (Tashian, 1989). The isoenzyme II is a high activity form, the structure of which is known (Liljas et al., 1972) and well refined (Eriksson, Jones & Liljas, 1988). The enzyme is inhibited by a number of inorganic ions as well as by sulfonamides (Lindskog et al., 1984). Extensive spectroscopic investigations of Co 2÷- substituted carbonic anhydrases and crystallo- graphic studies of the native enzyme have indicated that the coordination of the metal varies depending on the pH and the type of ligand (Lindskog, 1963; Bertini & Luchinat, 1982; Eriksson, Jones & Liljas, 1988; Vidgren, Liljas & Walker, 1990). This is in agreement with EXAFS studies of the Co 2÷ enzyme (Yachandra, Powers & Spiro, 1983). The cobalt elec- tronic absorption spectrum of the enzyme when inhibited by thiocyanate is interpreted as originating from a penta-coordinated metal (Bertini & Luchinat, 1982). This has been confirmed by crystallographic 0108-7681/92/030281-05506.00 methods on the zinc enzyme (Eriksson, Kylsten, Jones & Liljas, 1988). In addition to the three histi- dine ligands the inhibitor occupies a fourth and a water molecule a fifth coordination position. We wanted to extend these studies of penta- coordinated zinc in carbonic anhydrase in order to further elucidate the structure and function of the enzyme. At the same time we wanted to test the potential of Laue crystallography to observe small details in electron density which is a prerequisite to time-resolved protein crystallography. In contrast to the spectroscopic observations of the Co 2÷ enzyme we have observed that the zinc ion of the crystallized enzyme is tetra-coordinated in complex with bisulfite as well as at pH 6.0 where a water molecule is situated at the fourth coordination site. Interestingly, EXAFS studies of Zn 2+ carbonic anhydrase found that the average coordination number seems independent of pH, or of inhibitor binding, and is judged to be four (Yachandra et al., 1983). Experimental Crystals of human carbonic anhydrase II with space group P21, cell parameters a = 42.7, b--41.7, c = 73.0~, fl = 104.6 °, were grown, complexed with Hg 2+, in 2.3M (NH4)2SO4 at pH 8.5. Soaking experiments with the crystals are summarized in Table 1. Diffraction data were collected at 293 K on experimental stations 9.6 and 9.7 at the Daresbury Synchrotron Radiation Source (SRS), using poly- chromatic (Laue) radiation in the interval 0.40-2.6 )~ on stations 9.6 and 9.7. Data were extended to a resolution of 2.2 A and were sampled with a com- pleteness of 55-70% (Table 2). The crystals were elongated plates in morphology and were mounted with the a axis approximately © 1992 International Union of Crystallography