1H nuclear magnetic resonance investigation of cobalt(II) substituted carbonic anhydrase

The structure of ClO4 and NO3 adducts of cobalt(II) substituted bovine carbonic anhydrase have been investigated through 1D NOE and 2D 1H nuclear magnetic resonance (NMR) spectroscopy. For the first time two-dimensional NMR techniques are applied to paramagnetic metalloproteins other than iron-containing proteins. Several active site signals have been assigned to specific protons on the grounds of their scalar and dipolar connectivities and T1 values. The experimental dipolar shifts for the protons belonging to noncoordinated residues have allowed the identification of a plausible orientation of the magnetic susceptibility tensor around the cobalt ion as well as of the magnitude and the anisotropy of the principal susceptibility values. In turn, a few more signals have been tentatively assigned on the grounds of their predicted dipolar shifts. The two inhibitor derivatives have a very similar orientation but a different magnitude of the chi tensor, and the protein structure around the active site is highly maintained. The results encourage a more extensive use of the two-dimensional techniques for obtaining selective structural information on the active site of metalloenzymes. With this information at hand, comparisons within homologous series of adducts with various inhibitors and/or mutants of the same enzyme of known structure should be possible using limited sets of NMR data

Structural and functional characterization of sperm whale myoglobin mutants: role of arginine (E10) in ligand stabilization.

H-1 NMR and ligand-binding data were used to assess the role of residue Arg(E10) in ligand stabilization of several site-directed mutants, all carrying the His(E7) to Val substitution, obtained using a synthetic sperm whale myoglobin gene. Arg(E10) was previously found to form a hydrogen bond with the ligand in fluoro-, azido- and cyanomet derivatives of Aplysia limacina myoglobin, which lacks the distal His(E7) [Qin, J., La Mar, G. N., Ascoli, F., Bolognesi, M., & Brunori, M. (1992) J. Mol. Biol. 224, 891-897]. NMR analysis of the paramagnetically induced relaxation, hyperfine shift patterns, and dipolar connectivities shows that Arg(E10) also falls into the distal pocket in the engineered sperm whale myoglobin mutants and resides at an H-bonding distance from the Fe3+-bound cyanide. The rate constant for cyanide dissociation from the ferrous derivative was determined by stopped-flow experiments; the ligand stabilization achieved by Arg(E10) is similar to that exerted by His(E7) in wild-type sperm whale myoglobin, and both are very different from the His(E7)Val single mutant. Contrary to that for the wild-type, the cyanide dissociation rate constant for the mutant containing Arg(E10) is essentially independent of pH (from 6 to 9), as expected on the basis of the guanidinium group of Arg having a pK > 10. This finding is consistent with the NMR data in which the chemical shift of the Arg(E10) N(epsilon)H is insensitive to pH (6-9), as is also observed in Aplysia limacina cyanometmyoglobin. Equilibrium and kinetic data on the binding of azide to ferric myoglobins also indicate a similar trend, confirming the significant stabilizing role of Arg(E10). At acid pH (5-6), none of the sperm whale myoglobin mutants lacking His(E7) have water coordinated to the heme Fe3+, as revealed by H-1 NMR and optical spectroscopy; this suggests that Arg(E10) is out of the distal pocket in acid ferric myoglobin. However, at alkaline pH, the formation of the alkaline ferric derivative is also stabilized by Arg(E10), as demonstrated by the pK values of the acid-alkaline optical transition. The results prove the previously proposed hypothesis that Arg(E10) has a stabilizing effect on anionic ligands bound to the heme iron [Bolognesi, M., Coda, A., Frigerio, F., Gatti, G., Ascenzi, P., & Brunori, M. (1990) J. Mol. Biol. 213, 621-625; Cutruzzola, F., Travaglini Allocatelli, C., Ascenzi, P., Bolognesi, M., Sligar, S. G., & Brunori, M. (1991) FEBS Lett. 282, 281-284]. In addition, our study indicates that the side chain at position CD3 has no significant effect in the control of ligand stabilization, since similar kinetic and NMR properties have been observed in triple mutants bearing an extra mutation at position CD3