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Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the beta-CH2 protons of the eight metal coordinated cysteines. Geminal connectivities of Cys beta-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding alpha-CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed.

600-MHz 1H-NMR and nuclear Overhauser enhancement spectroscopy (NOESY) spectra in 2H2O and H2O, as well as truncated driven NOE difference spectra in H2O of reduced human Cu(II)2Zn(II)2 superoxide dismutase (Cu/Zn SOD) have been recorded and used to assign the active-site proton signals. A derivative with histidines selectively deuteriated in the C2 position has been used for the detection of the HC2 histidine protons, 16 out of 17 observed signals of the 18 active-site histidine ring protons have been assigned. The results are compared with previous proposals based on more limited data sets. The numerous cross peaks confirm that the structure in solution is essentially similar to the crystallographic data obtained on the oxidized form. Probably this holds also for His63 which in the reduced form is not bridging any more the two metal ions. The effects of azide binding on the exchangeable 1H-NMR signals of the reduced protein are also reported.

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through 1H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.

The structure-function relationship in Cu,Zn superoxide dismutase has been partially elucidated by the combined use of many spectroscopic techniques (electronic spectroscopy, circular dichroism, EPR and NMR) and site-directed mutagenesis techniques. The comparison of the spectroscopic and catalytic properties of various mutants, in which some active site residues have been substituted through site-directed mutagenesis, allowed us to establish that the activity is in general more sensitive to electrostatic effects rather than to steric effects or changes in the copper hydration or coordination geometry.

Truncated, steady state and transient NOE experiments have been performed on bovine Cu2Co2 superoxide dismutase. The effectiveness of the different NOE experiments in the general case of paramagnetic macromolecules is discussed. It is concluded that steady state NOEs give superior results. The validity of the two spins approximation is discussed, and NOE values for a fully coupled set of nuclei have been calculated. Transient NOE experiments, when properly performed, confirm the previous assignment of the hyperfine shifted signals in Cu2Co2SOD based on steady state NOE measurements [(1989) Inorg. Chem. 28, 4650] and eliminate any further reason for controversy on an important issue as the assignment of the 1H NMR signals of protons of metal-coordinated imidazoles.

The epr spectra at 4.2 K of inhibitor derivatives of cobalt carbonic anhydrase have been recorded. The spectra can be grouped into two classes according to whether the low-field signal is broad or sharp and with g ranges of 6.1-6.8, 2.3-2.9, 1.6-1.8, and 5.8-6.2, 2.2-2.8, 1.5-1.8, respectively, The two kinds of spectra have been empirically related to the features of the room-temperature solution electronic spectra. A third kind of epr spectrum with a single broad signal is obtained when the inhibitor is in large excess. The possibility of using the epr spectra for deducing the geometry of cobalt enzymes is discussed.

The 1H NMR spectra of the cyanide adduct of Cu2Co2-superoxide dismutase have been remeasured at pH 7.5. The exchange rate of CN- is slow on the NMR time scale. The correlation with the spectrum of the unligated enzyme has been established through saturation-transfer techniques of the system in which 50% of the cyanide adduct is formed and through comparison with the spectrum of a Cu2Co2-superoxide dismutase-CN- sample in which the histidines have been deuterium labeled at the position epsilon 1. The similarities between the spectra of the CN- and N-3 derivatives are stressed, in particular with respect to the removal from copper coordination of the same histidine, assigned as His-46.

1H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition 13C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the beta-CH2 protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The 1H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3- and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD+ only the resonances of the beta-CH2 protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper(II) substituted human and bovine carbonic anhydrases B in the presence of bicarbonate have been investigated in solution through water-solvent proton nuclear magnetic resonance (nmr) at variable magnetic fields. HCO3-, contrary to all the other monoanionic inhibitors, partially reduces the water proton relaxation rates. This has been accounted for on the basis of the availability within the active cavity of two coordination positions partially overlapping. 13C-nmr measurements on both CO2 and HCO3- confirm that HCO3- binds the metal, whereas CO2 interacts with the paramagnetic center at nonbonding distance. The upper limit for the CO2 in equilibrium HCO3- interconversion has been estimated to be 10 sec-1.

The bis and mono cobalt(II) derivatives of conalbumin in the presence of bicarbonate have been prepared. The 1H NMR spectra have been recorded at 60 MHz. Well-shaped isotropically shifted signals in the range -100 to +100ppm have been observed for both the mono and bis cobalt(II) derivatives; besides the intensity, all the isotropically shifted signals for the two derivatives are superimposable, indicating that the two sites are so similar as to be indistinguishable within the resolution of the technique. With the aid of T1 measurements, the signals have been assigned to the protons of two histidine and two tyrosinate ligands. The spectra are consistent with six-coordinated, high-spin cobalt(II) chromophores with two trans tyrosinate moieties at each site.

Cobalt(II) ovotransferrin bicarbonate and oxalate ternary complexes were prepared and investigated in the pH range 7-10.5. Cobalt(II) provides an excellent and unique spectroscopic probe to monitor subtle structural differences in solution between the two sites of ovotransferrin and to investigate the structural dependence on pH. CD spectroscopy on one side and 1H NMR spectroscopy of isotropically shifted signals on the other are extremely sensitive techniques and are particularly suited for high spin cobalt(II)-containing compounds. In the case of the oxalate derivative the metal-binding ability of the protein is different at the two binding sites and is pH dependent; the CD spectra reveal two different sites, one of which is clearly pH dependent with a pKa of 9.5. On the contrary the bicarbonate analogue does not show any spectral difference between the two sites; both of them change with pH, the pKa being again 9.5. 1H NMR spectra of the oxalate derivatives at pH 7-8 reveal the presence of conformers, the distribution of which depends on the H2O/D2O ratio. Such conformers are not revealed in the bicarbonate system; at pH around 10 the NMR spectra of both systems show inequivalence between the two sites and/or the presence of different conformers for each site. Such differences are discussed in terms of the possible implications in mechanism and function. The overall spectral data are consistent with the donor groups being two histidines, two tyrosines, the synergistic anion, and possibly a solvent molecule.

The interactions between hydrated trichloroacetaldehyde and cobalt(II)bovine carbonic anhydrase B have been investigated as a function of pH by means of electronic spectroscopy of FT nmr spectroscopy. The hydrated aldehyde is bound to the metal ion and its apparent affinity constant is pH dependent with a bell-shaped profile. The kinetic parameters of the dissociation process have also been determined.

The preparation procedure of vanadium(III) transferrin and its stability are confirmed to be as previously reported. The electronic spectra of vanadium(III), oxovanadium(IV), and vanadium(V) transferrin derivatives are comparatively discussed. A band in the near infrared of the oxovanadium(IV) derivative is observed for the first time.

The visible spectra of solutions of cobalt(II) bovine carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) with increasing amounts of acetate have allowed the determination of the apparent inhibition constants and of the extrapolated limit spectra of the completely bound enzyme. The electronic spectra have been interpreted on the basis of the presence of five coordinate adduct species. 13C and 1H NMR spectra have also been recorded and discussed on the basis of the metal enzyme-acetate type of interactions. Titrations by means of NMR spectroscopy of the acetate-cobalt(II) bovine carbonic anhydrase with p-toluenesulfonamide and azide indicate the existence of two binding sites for the acetate group.

The affinity of bicarboxylate ions (from oxalate to glutarate) for cobalt (II) bovine carbonic anhydrase has been investigated and compared with that of acetate and propionate. The oxalate ion shows a much greater affinity for the enzyme than acetate, whereas the other bicarboxylate ions have very little tendency to bind the enzyme. In every case, and particularly for the oxalate, the apparent affinity constants dramatically increase with decreasing pH. On the basis of the electronic spectra a five-coordinate structure is proposed for all of the above derivatives. Carbon-13 NMR data have been discussed in terms of the oxalate ion chelating the metal ion and/or interacting with the wall of the active cavity.

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N3-, NCO-, and NCS-) has been studied by 1H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E.I, E.I2, and E.I.X, each with characteristic NMR features, have been identified. Thus, for example, L-Phe binds close to the metal ion to form a 1:1 complex, whereas D-Phe binds stepwise, first to a nonmetal site and then to the metal ion to form a 2:1 complex. Both acetate and phenylacetate also form 2:1 adducts stepwise with the enzyme, but beta-phenylpropionate gives a 2:1 complex without any detectable 1:1 intermediate. N3-, NCO-, and NCS- generate E.I.X ternary complexes directly with Co(II)CPD.L-Phe and indirectly with the D-Phe and carboxylate inhibitor 2:1 complexes by displacing the second moiety from its metal binding site. The NMR data suggest that when the carboxylate group of a substrate or inhibitor binds at the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The 1H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

13C NMR T1 and T2 measurements have been performed on cobalt(II) substituted carboxypeptidase A in the presence of carboxylate-13C-enriched L- and D-phenylalanine. Upon binding to the cobalt enzyme, the longitudinal and transverse relaxation rates T1p-1 and T2p-1 of these inhibitors are enhanced significantly compared to the zinc enzyme, allowing both determination of an affinity constant for inhibitor binding, K, and calculation of the metal-13C carboxylate distances. The L-and D- Phe concentration dependence of T2p-1 yields affinity constants of 290 +/- 60M-1 and 670 +/- 90M-1. The distance measurements calculated for Co-13C from T1p-1 are 0.39 +/- 0.04 and 0.42 +/- 0.04 nm for L-Phe and D-Phe. Both values are too great for direct coordination of their carboxylate groups to the metal atom. Upon formation of their respective ternary enzyme.Phe.N3- complexes, the distances are essentially unaltered. In conjunction with electronic absorption studies on these complexes it can be concluded that N3-, but not the amino acid carboxylate, is bound to the metal.

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.

It is known that H2O2 at pH 10, inactivates copper(II)-zinc(II)-SOD although not much information is available on what happens at the ligands coordinated to the two metal ions. We have reinvestigated the system through the electronic and 1H NMR spectra of the cobalt(II) and copper(II)-cobalt(II) derivatives. Such studies indicate that the coordinated residues are maintained although there is evidence of some flexibility of the donor groups. The coordination around copper is slightly more tetragonal. Azide binding to the copper ion does not cause the complete detachment of one of the histidines from the copper coordination sphere, as happens with the untreated enzyme.

We have measured the paramagnetic contribution to the magnetic relaxation rate of solvent protons in highly purified, buffer- and salt-free solutions of Co(2+)-substituted human carbonic anhydrase B (HCAB), as a function of pH in the range 5.5-10 and as a function of magnetic field. We have also measured the optical absorption at 640 nm to characterize the enzyme. The relaxation rates vary with pH much as does the CO(2) hydration activity, increasing with increasing pH. We find that the relaxation rates at all intermediate values of pH can be described as linear combinations of the rates obtained at the extremes of pH used, indicating the existence of low- and high-pH forms of the enzyme with pH-dependent concentrations. The optical data can be similarly represented. The fraction of high-pH form present, determined from either the relaxation or optical data, has a pK(a) of approximately 7.6 when approximated by a single ionization. The data are very similar to that for HCAB in the presence of buffer, in contrast to the bovine enzyme for which the pK(a) is affected substantially by the presence of sulfate. Previous analysis of the high relaxation rates at high pH indicated rapid exchange of Co(2+)-liganded protons, possible only if these exchanging protons were conveyed by water molecules. On the other hand, the present demonstration of the existence of two forms of HCAB in highly purified solutions, coupled with other data, argues strongly for ionization of a water molecule ligand of the metal ion at the active site, with OH(-) as the solvent-donated ligand at high pH. We propose a mechanism of ligand exchange at high pH that reconciles these ostensibly conflicting requirements by invoking a pentacoordinate intermediate having both OH(-) and H(2)O as ligands. Proton exchange can be rapid between these ligands because charge transfer without net ionization can occur, so that the leaving water can carry away the initial OH(-). The low-pH form is a thermal mixture of tetra- and pentacoordinate species, the latter having low relaxation rates by analogy with inhibitor derivatives of the enzyme and model systems. The proposed associative ligand-exchange mechanism reconciles the distinctions between the OH- and H(2)O-models of carbonic anhydrase by merging them, providing the first model is consistent with the observed pH dependence of hydration activity, optical absorption, and solvent magnetic relaxation.

The uptake of cobalt(II) ions by apoalkaline phosphatase at pH 8 (the pH optimum for activity) has been investigated by the combined use of electronic and 1H NMR spectroscopies. The presence of fast-relaxing high spin cobalt(II) ions in the active site cavity of the protein induces sizable isotropic shifts of the 1H NMR signals of metal-coordinated protein residues, allowing us to propose a metal uptake pattern by the various metal binding sites both in the presence and in the absence of magnesium ions. In the absence of magnesium the active site is not organized in specific metal binding sites. The first equivalent of cobalt(II) ions per dimer binds in an essentially unspecific and possibly fluxional fashion, giving rise to a six-coordinated chromophore. The second and third equivalents induce the formation of increasing amounts of metal ions pairs, cooperatively arranged into the A and B sites of the same subunit with a five- and six-coordinated geometry, respectively. The fourth and fifth equivalents induce the formation of fully blocked A-B pairs in both subunits. Magnesium shows the property of organizing the metal binding sites, probably through coordination to the C sites. Electronic and 1H NMR titration with Co2+ ions show that the initial amount of fluxional cobalt is smaller than in the absence of magnesium and that A-B pairs are more readily formed. Titration of fully metalated Co4Mg2alkaline phosphatase samples with phosphate confirms binding of only one phosphate per dimer.

Water 1H-nuclear-magnetic-relaxation-dispersion (NMRD) measurements of solutions of several copper/zinc superoxide dismutase isoenzymes as well as mutants of the human isoenzyme have been performed in order to monitor the presence of exchangeable water at the copper(II) center. The results have been compared with other spectroscopic features of the various derivatives and with their catalytic efficiency. The decrease in the amount of water in the first coordination sphere, detected through NMRD, parallels, in most cases, the increase in the tetragonal nature of the copper ion. On the other hand, the catalytic activity seems unrelated to the presence of water. Most strikingly, the Ile137 mutant of the human isoenzyme, approximately equal to 90% active, has no water in the copper coordination sphere; this is taken as evidence that the electron transfer is not a water-mediated process. The variation in the pH dependence of NMRD data between the wild-type enzyme and the human Ile137 mutant has been found to parallel the variation in the pH dependence of activity.

Measurements of the magnetic field dependence of the longitudinal nuclear magnetic relaxation rates of solvent protons (NMRD profiles) in solutions of paramagnetic proteins have contributed significantly to the elucidation of the physical biochemistry of a number of metalloprotein systems. In many cases, NMRD profiles were used as indicators of chemical state, both static and dynamic [cf. Brewer, C. F., Brown, R. D., III, & Koenig, S. H. (1983) J. Biomol. Struct. Dyn. 1, 961-997], in part because a proper theoretical description of the data, with realistic assumptions for a model system, was computationally intractable. This has been particularly true for Cu2+-protein complexes, attributable in part to the S = 1/2 ground-state configuration of the Cu2+ ions; significant progress in interpreting such data has been made only recently [Bertini, I., Briganti, F., Luchinat, C., Mancini, M., & Spina, G. (1985) J. Magn. Reson. 63, 41-55]. We report NMRD profiles for solutions of Cu2+ - and VO2+-substituted human transferrin, both S = 1/2 ions, as well as computations that include the effects of the anisotropic hyperfine interactions of the paramagnetic ions with their respective nuclei. The description of the data that results from these computations is quite good, sufficiently so that one can say with confidence that the protons that contribute to the relaxation are rather distant (approximately 3.5 A) from the ions and in rapid exchange (approximately 10(8) s-1) with solvent. A possible view, consistent with what is known of the biochemistry of these substituted transferrins, is that relaxation occurs in the second coordination sphere: the exchanging entity is a water molecule hydrogen bonded to a donor atom of the metal ion complex.

The electronic and 1H NMR spectra are reported for the cobalt(II) alkaline phosphatase (EC 3.1.3.1.) system at pH around 6 in the range 0-2 mol of cobalt per mol protein. It is shown that under the present experimental conditions cobalt(II) selectively populates the A sites. Three isotropically shifted NH signals have been detected in the A site that indicate the presence of three histidines in the coordination sphere of cobalt(II). The electronic spectra and the nuclear relaxation properties are consistent with pentacoordination of cobalt(II) in the A site. The finding of reproducible preparation routes for the derivatives, and of appropriate experimental conditions for the observation of their 1H NMR spectra, open new possibilities for the spectroscopic investigation of alkaline phosphatase.

The results of a study on the interaction between cobalt(II) bovine carbonic anhydrase and the alpha-amino acids L(+) and D(-)alanine, glycine and betaine are reported. L(+)alanine and glycine have been found to have larger affinity for the enzyme than D(-)alanine whereas no sizable affinity is shown by betaine. The electronic spectra indicate that these systems are similar to that containing the acetate ion. Utilizing the inhibition properties of L(+)alanine at various pH an analysis of the species involved in the inhibition reaction is presented.