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.

Proton nuclear Overhauser effect study of the heme active site structure of Coprinus macrorhizus peroxidase.

Proton nuclear Overhauser effect and paramagnetic relaxation measurements have been used to define more extensively the heme active site structure of Coprinus macrorhizus peroxidase, CMP (previously known as Coprinus cinereus peroxidase), as the ferric low-spin cyanide ligated complex. The results are compared with other well-characterized peroxidase enzymes. The NMR spectrum of CMPCN shows changes in the paramagnetically shifted resonances as a function of time, suggesting a significant heme disorder for CMP. The presence of proximal and distal histidine amino acid residues are common to the heme environments of both CMPCN and HRPCN. However, the upfield distal arginine signals of HRPCN are not evident in the 1H-NMR spectra of CMPCN.

Proton nuclear Overhauser effect study of the heme active site structure of chloroperoxidase.

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C epsilon-H proton of the distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J.H., Hall, K., & Hager, L.P. (1986) Biochemistry 25, 347-356].

1H-NMR characterization of cucumber peroxidases.

Two peroxidase isoenzymes from Cucumber seedlings, one acidic (pI = 4) and one basic (pI = 9), were characterized by 1H-NMR spectroscopy. The NMR spectra were obtained in the native (ferric high-spin) and cyanide ligated (ferric low-spin) forms of both isoenzymes. The NMR spectral comparison of paramagnetically shifted resonances with those of the well characterized horseradish peroxidase C, HRP(C), isoenzyme indicates that both cucumber peroxidases have a protohemin IX prosthetic group with proximal histidine coordinated to the heme iron. The downfield heme 1H-NMR shift pattern is distinct for each isoenzyme, and this reflects presumably dissimilar heme active site environments. The basic isoenzyme shows less asymmetry in heme 1H-NMR signals as compared to the acidic isoenzyme or HRP(C) isoenzyme. It was also found that the acidic cucumber peroxidase exists predominantly as a monomeric species in solution with 30 kDa molecular mass as opposed to its earlier characterization as a 60 kDa dimeric protein.

Proton NMR study of the comparative electronic/magnetic properties and dynamics of the acid in equilibrium with alkaline transition in a series of ferricytochromes c'.

The proton NMR spectra of ferricytochrome c' from Rhodopseudomonas palustris, Rhodospirillum molischianum, Rhodospirillum rubrum, and Chromatium vinosum have been investigated for the purpose of further elucidating the common spectral and/or structural properties for this subclass of cytochromes in the acidic and alkaline forms, and to characterize in detail the dynamics and structural basis for this acid in equilibrium with alkaline transition. The identification of strongly upfield-shifted meso-H peaks in all but C. vinosum ferricytochrome c' at weakly acidic to neutral pH is consistent with, but not proof for, S = 3/2 character for the spin state of C. vinosum, but argues for primarily S = 5/2 character for the other three proteins. Hence, we conclude that the quantum mechanically mixed S = 3/2, S = 5/2 spin ground state of neutral pH C. vinosum ferricytochrome c' is an anomaly rather than a characteristic of this class of proteins. The 1H NMR spectra of ferricytochromes c' at alkaline pH again exhibit strong similarities among all members except that for C. vinosum. Two pK values are observed for ferricytochrome c' for R. molischianum and C. vinosum, of which the higher value pK is accompanied by significant line broadening, as found earlier for the proteins from both R. rubrum and R. palustris. Detailed analysis of the exchange line broadening for all four proteins reveals that hydrolysis is the rate-limiting step, with base catalysis occurring at about the same rate in the diffusion control limit for all four proteins. The variable first order dissociation rates of the alkaline species reveal differential stabilities of that species in the order R. palustris greater than R. molischianum greater than R. rubrum much greater than C. vinosum. The rates of exchange of the axial His imidazole labile proton was determined by linewidth and saturation transfer analysis and shown to occur via base catalysis at the same diffusion control rate as found for the acid----alkaline transition for the oxidized protein, and support the proposal that the acid----alkaline transition involves simply the abstraction of a proton from the neutral His imidazole to yield an imidazolate.

A nuclear Overhauser effect study of the active site of myeloperoxidase. Structural similarity of the prosthetic group to that on lactoperoxidase.

The low-spin, cyanide-ligated ferric complex of the intact bovine granulocyte myeloperoxidase (MPO-CN) has been studied by proton nuclear magnetic resonance utilizing the nuclear Overhauser effect (NOE). This is the largest globular protein (approximately 1.5 x 10(5) for the intact alpha 2 beta 2 tetrameric species) for which successful NOEs have been observed without serious interference of spin diffusion, and demonstrably confirms the utility of such studies on large paramagnetic as compared to diamagnetic proteins. The 1H NMR spectrum of MPO-CN is found to have a remarkable similarity in the number, resonance pattern, and metal ion-induced relaxation properties of the resolved, hyperfine-shifted resonances to those reported earlier for the analogous complex of bovine lactoperoxidase (LPO-CN); moreover, the interproton connectivities between pairs of hyperfine-shifted proton sets, as reflected by the NOEs, are also essentially the same (Thanabal, V., and La Mar, G. N. (1989) Biochemistry 28, 7038-7044). Since the extracted prosthetic group of lactoperoxidase is a porphyrin with proposed functionalization of the 8-methylene group (Nichol, A. W., Angel, L. A., Moon, T., and Clezy, P. S. (1987) Biochem. J. 247, 147-150), we interpret the resultant similarity in 1H NMR spectral parameters for LPO-CN and MPO-CN as indicating that the prosthetic groups in MPO and LPO are very similar, and hence likely both porphyrins with a similarly functionalized periphery that allows covalent linkage to the protein matrix. The hyperfine shift pattern of the broadest resolved lines lead to their assignment to the axial histidyl imidazole side chain. Two pairs of resonances are found to have similar relaxation properties and/or dipolar as similarly shifted resonances that arise from a distal His and Arg in horseradish peroxidase (as also found in LPO-CN), and suggest that MPO also possesses these catalytically functional residues in the distal heme pocket.

Identification of localized redox states in plant-type two-iron ferredoxins using the nuclear Overhauser effect.

The homonuclear Overhauser effect (NOE), in conjunction with nonselective spin-lattice relaxation measurements, has been employed to assign the contact-shifted resonances for the reduced form of two typical plant-type two-iron ferredoxins from the algae Spirulina platensis and Porphyra umbilicalis. These results demonstrate that the NOE should have broad general applicability for the assignments and electronic structural elucidation of diverse subclasses of paramagnetic iron-sulfur cluster proteins. NOE connectivities were detected only among sets of resonance exhibiting characteristically different deviations from Curie behavior, providing strong support for the applicability of the spin Hamiltonian formulation for the NMR properties of the antiferromagnetically coupled iron clusters [Dunham, W. R., Palmer, G., Sands, R. H., & Bearden, A. J. (1971) Biochim. Biophys. Acta 253, 373-384; Banci, L., Bertini, I., & Luchinat, C. (1989) Struct. Bonding (in press)]. The geminal beta-methylene protons for the two cysteines bound to the iron(II) center were clearly identified, as well as the C alpha H and one C beta H for each of the cysteines bound to the iron(III). The identification of the iron bound to cysteines 41 and 46 as the iron(II) in the reduced protein was effected on the basis of dipolar contacts between the bound cysteines, as predicted by crystal coordinates of S. platensis Fd [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. (Tokyo) 90, 1763-1773].(ABSTRACT TRUNCATED AT 250 WORDS)

NMR studies of carbonic anhydrase-fluorinated benzenesulfonamide complexes.

Fluorine NMR has been used to examine complexes formed by 2-fluoro-, 3-fluoro-, and 2,5-difluorobenzenesulfonamide and human carbonic anhydrases I and II. The results show that all three inhibitors form complexes with both isozymes that have 2:1 inhibitor/enzyme stoichiometry. The fluorine spectra observed for all inhibitor-isozyme combinations are consistent either with rapid rotation of the aromatic ring of the inhibitor in the complexes or with preferential binding of only one of the two possible conformations of the inhibitors that are isomeric by virtue of rotation about the C1-C4 bond of the fluoro aromatic ring. Because ring rotation is slow in the case of the pentafluorobenzenesulfonamide-CA I complex, selective binding of rotamers is the explanation of these observations presently favored. A computer graphics study shows that formation of 2:1 complexes of CA I is feasible without appreciable distortion of the protein tertiary structure found in the crystalline state.

NMR studies of carbonic anhydrase-4-fluorobenzenesulfonamide complexes.

Binding of 4-fluorobenzenesulfonamide to human carbonic anhydrases I and II has been studied by proton, fluorine, and nitrogen-15 nuclear magnetic resonance spectroscopy. All three types of experiments provide evidence that the stoichiometry of the interaction of this inhibitor with both enzymes is 2 mol of inhibitor bound per mole of enzyme. Observations which suggest that the bound forms are involved in an exchange process that is rapid at room temperature but slower at 2 degrees C are described. Nitrogen-15 shift data show that the bound inhibitors are present at the active site as anions. The proton experiments indicate appreciable reorganization of the tertiary structure of the protein upon binding. Saturation-transfer experiments to determine the rate of dissociation of the inhibitor-enzyme complex lead to the conclusion that the dissociation process is more complicated than a simple free-bound equilibrium.