Ligation Geometry Research Articles

Role of Calcium in Metalloenzymes: Effects of Calcium Removal on the Axial Ligation Geometry and Magnetic Properties of the Catalytic Diheme Center in MauG

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and Mössbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and Mössbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.

Prediction of structures of zinc‐binding proteins through explicit modeling of metal coordination geometry

Metal ions play an essential role in stabilizing protein structures and contributing to protein function. Ions such as zinc have well-defined coordination geometries, but it has not been easy to take advantage of this knowledge in protein structure prediction efforts. Here, we present a computational method to predict structures of zinc-binding proteins given knowledge of the positions of zinc-coordinating residues in the amino acid sequence. The method takes advantage of the "atom-tree" representation of molecular systems and modular architecture of the Rosetta3 software suite to incorporate explicit metal ion coordination geometry into previously developed de novo prediction and loop modeling protocols. Zinc cofactors are tethered to their interacting residues based on coordination geometries observed in natural zinc-binding proteins. The incorporation of explicit zinc atoms and their coordination geometry in both de novo structure prediction and loop modeling significantly improves sampling near the native conformation. The method can be readily extended to predict protein structures bound to other metal and/or small chemical cofactors with well-defined coordination or ligation geometry.

Bistable Molecular Switches Based on Linkage Isomerization in Ruthenium Polypyridyl Complexes with a Ligand‐Bound Ambidentate Motif

Electron-transfer-induced linkage isomerization was investigated in a series of bis-tridentate Ru polypyridyl complexes [Ru(L-X-OH)(Y-tpy)](2+) with ambidentate ligand L-X-OH=bpy-C(R)(OH)-py (bpy=2,2'-bipyridine; py=pyridine; R=H, Me, Ph, or tBu) and spectator ligand Y-tpy (tpy=2,2':6',2''-terpyridine; Y=p-tolyl, p-PhCO(2)Me, Cl, OEt, N-pyrrolidine). The ligand-bound ambidentate motif switches reversibly between N and O coordination in the Ru(II) and Ru(III) state, respectively. The potentials of the Ru(III/II) couple differ by about 0.5 V between the isomers, and this results in a bistable electrochemical response of the molecular switches. The effects of structural modifications in form of substituents on the linking carbon atom of the ambidentate ligand and on the central pyridine moiety of the spectator ligand were investigated by electrochemical and computational methods. Differences in isomerization behavior span six orders of magnitude in rate constants and two orders of magnitude in equilibrium constants. The results can be interpreted in terms of steric and electronic substituent effects and their influence on rotational barriers, ligation geometry, and electron deficiency of the metal center.

Correlation of Rhombic Distortion of the Type 1 Copper Site of M98Q Amicyanin with Increased Electron Transfer Reorganization Energy

Mutation of the axial Met ligand of the type 1 copper site of amicyanin to Ala or Gln yielded M98A amicyanin, which exhibits typical axial type 1 ligation geometry but with a water molecule providing the axial ligand, and M98Q amicyanin, which exhibits significant rhombic distortion of the type 1 site (Carrell, C. J., Ma, J. K., Antholine, W. E., Hosler, J. P., Mathews, F. S., and Davidson, V. L. (2007) Biochemistry 46, 1900-1912). Despite the change of the axial ligand, the M98Q and M98A mutations had little effect on the redox potential of copper. The true electron transfer (ET) reactions from O-quinol methylamine dehydrogenase to oxidized native and mutant amicyanins revealed that the M98A mutation had little effect on kET, but the M98Q mutation reduced kET 45-fold. Thermodynamic analysis of the latter showed that the decrease in kET was due to an increase of 0.4 eV in the reorganization energy (lambda) associated with the ET reaction to M98Q amicyanin. No change in the experimentally determined electronic coupling or ET distance was observed, confirming that the mutation had not altered the rate-determining step for ET and that this was still a true ET reaction. The basis for the increased lambda is not the nature of the atom that provides the axial ligand because each uses an oxygen from Gln in M98Q amicyanin and from water in M98A amicyanin. Comparisons of the distance of the axial copper ligand from the equatorial plane that is formed by the other three copper ligands in isomorphous crystals of native and mutant amicyanins at atomic resolution indicate an increase in distance from 0.20 A in the native to 0.42 A in M98Q amicyanin and a slight decrease in distance for M98A amicyanin. This correlates with the rhombic distortion caused by the M98Q mutation that is clearly evident in the EPR and visible absorption spectra of the protein and suggests that the extent of rhombicity of the type 1 copper site influences the magnitude of lambda.

Generation of Novel Copper Sites by Mutation of the Axial Ligand of Amicyanin. Atomic Resolution Structures and Spectroscopic Properties,

Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (<or=1.0 A). The crystal structure of oxidized M98A amicyanin exhibits a type 1 ligation geometry but with the axial ligand provided by a water, which fills the void left by the mutation of Met to Ala. The protein undergoes a reversible switch in ligation geometry when going from the aqueous to the frozen state. The visible absorption spectrum in solution is characteristic of type 1 copper, consistent with the crystal structure. On freezing, the blue color is lost, and EPR spectroscopy reveals that the copper is primarily type 2. The crystal structure of reduced M98A amicyanin exhibits an unprecedented ligation geometry in which the His95-Cu coordination is broken, and copper is left with only two ligands from His53 and Cys92 in an almost linear coordination. The replacement of Met98 with Gln yielded a type 1 copper site with increased rhombicity evident from its EPR and visible absorption spectra, and an increase in distance from Cu to the trigonal equatorial plane seen in the crystal structure. Gln98 coordinates more strongly with copper than Met, and the oxidized and reduced forms each exhibit two alternate conformers. EPR and metal analysis of oxidized M98Q amicyanin indicate that a small population of the protein contains weakly bound type 2 copper, which may be removed by washing with EDTA. These results demonstrate that the identity as well as position and rigidity of the axial ligand of the type 1 copper site has a profound influence in the uptake specificity of metal ions, protein stability, and determination of the active site geometry and its spectroscopic properties.

High-Resolution Crystal Structure of Manganese Peroxidase: Substrate and Inhibitor Complexes,

Manganese peroxidase (MnP) is an extracellular heme enzyme that catalyzes the peroxide-dependent oxidation of Mn(II) to Mn(III). The Mn(III) is released from the enzyme in complex with oxalate. One heme propionate and the side chains of Glu35, Glu39, and Asp179 were identified as Mn(II) ligands in the 2.0 A resolution crystal structure. The new 1.45 A crystal structure of MnP complexed with Mn(II) provides a more accurate view of the Mn-binding site. New features include possible partial protonation of Glu39 in the Mn-binding site and glycosylation at Ser336. This is also the first report of MnP-inhibitor complex structures. At the Mn-binding site, divalent Cd(II) exhibits octahedral, hexacoordinate ligation geometry similar to that of Mn(II). Cd(II) also binds to a putative second weak metal-binding site with tetrahedral geometry at the C-terminus of the protein. Unlike that for Mn(II) and Cd(II), coordination of trivalent Sm(III) at the Mn-binding site is octacoordinate. Sm(III) was removed from a MnP-Sm(III) crystal by soaking the crystal in oxalate and then reintroduced into the binding site. Thus, direct comparisons of Sm(III)-bound and metal-free structures were made using the same crystal. No ternary complex was observed upon incubation with oxalate. The reversible binding of Sm(III) may be a useful model for the reversible binding of Mn(III) to the enzyme, which is too unstable to allow similar examination.

Structure of Acinetobacter strain ADP1 protocatechuate 3, 4-dioxygenase at 2.2 A resolution: implications for the mechanism of an intradiol dioxygenase.

The crystal structures of protocatechuate 3,4-dioxygenase from the soil bacteria Acinetobacterstrain ADP1 (Ac 3,4-PCD) have been determined in space group I23 at pH 8.5 and 5.75. In addition, the structures of Ac 3,4-PCD complexed with its substrate 3, 4-dihydroxybenzoic acid (PCA), the inhibitor 4-nitrocatechol (4-NC), or cyanide (CN(-)) have been solved using native phases. The overall tertiary and quaternary structures of Ac 3,4-PCD are similar to those of the same enzyme from Pseudomonas putida[Ohlendorf et al. (1994) J. Mol. Biol. 244, 586-608]. At pH 8.5, the catalytic non-heme Fe(3+) is coordinated by two axial ligands, Tyr447(OH) (147beta) and His460(N)(epsilon)(2) (160beta), and three equatorial ligands, Tyr408(OH) (108beta), His462(N)(epsilon)(2) (162beta), and a hydroxide ion (d(Fe-OH) = 1.91 A) in a distorted bipyramidal geometry. At pH 5.75, difference maps suggest a sulfate binds to the Fe(3+) in an equatorial position and the hydroxide is shifted [d(Fe-OH) = 2.3 A] yielding octahedral geometry for the active site Fe(3+). This change in ligation geometry is concomitant with a shift in the optical absorbance spectrum of the enzyme from lambda(max) = 450 nm to lambda(max) = 520 nm. Binding of substrate or 4-NC to the Fe(3+) is bidentate with the axial ligand Tyr447(OH) (147beta) dissociating. The structure of the 4-NC complex supports the view that resonance delocalization of the positive character of the nitrogen prevents substrate activation. The cyanide complex confirms previous work that protocatechuate 3,4-dioxygenases have three coordination sites available for binding by exogenous substrates. A significant conformational change extending away from the active site is seen in all structures when compared to the native enzyme at pH 8.5. This conformational change is discussed in its relevance to enhancing catalysis in protocatechuate 3,4-dioxygenases.

EPR properties of mixed-valent mu-oxo and mu-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K.

Radiolytic reduction at 77 K of oxo/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S = 1/2 ground state with small g-anisotropy as characterized by the uniaxial component (gz-gav/2 < 0.03) observable at temperatures as high as approximately 100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (gz-gav/2 > 0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins.

Substrate and substrate analog binding to endothelial nitric oxide synthase: electron paramagnetic resonance as an isoform-specific probe of the binding mode of substrate analogs.

The binding of arginine analogs to endothelial nitric oxide synthase (eNOS, NOSIII) perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Using electron paramagnetic resonance as a probe of heme ligation geometry, four categories of high-spin complex could be distinguished. These are analogous to the four classes of high-spin complexes, stabilized individually by the binding of L-arginine, N-hydroxy-L-arginine (NHA), N-methyl-L-arginine (NMA), and N-nitro-L-arginine (NNA), which we have previously reported for the other two isoforms. Each of these species is five-coordinate and retains the axial thiolate ligand but each differs in its ligation geometry. N-Methyl-L-arginine is a relatively poor inhibitor of eNOS, and the NMA complex of eNOS differs from the N-methyl-L-arginine complexes of inducible nitric oxide synthase (iNOS, NOSII) and neuronal nitric oxide synthase (nNOS, NOSI) in that it is of lower rhombicity. We previously showed that inducible nitric oxide synthase, which binds NNA less tightly than eNOS and nNOS, could not form the lower rhombicity NNA complex characteristic of nNOS. Endothelial nitric oxide synthase readily forms such lower rhombicity complexes, which correlates with the tight binding of NNA to this isoform. Arginine and tetrahydrobiopterin promote loss of the flavin free radical EPR signal, while arginine analog inhibitors stabilize the radical; this suggests that the residual flavin radicals can serve as a source of reducing equivalents for slow turnover in the absence of endogenous reductant.

Electron paramagnetic resonance spectroscopy of the heme domain of inducible nitric oxide synthase: binding of ligands at the arginine site induces changes in the heme ligation geometry.

The electron paramagnetic resonance spectra of the heme domain of inducible nitric oxide synthase (iNOS) demonstrate a close relationship to the corresponding spectra of the neuronal isoform (nNOS). The binding of ligands to the iNOS arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. The iNOS forms five-coordinate, high-spin complexes with arginine analogs which are clearly related to the corresponding complexes of nNOS. Studies indicate that the binding of L-arginine, N(omega)-hydroxy-L-arginine (NHA), and N(omega)-methyl-L-arginine (NMA) produces various spectroscopic species closely corresponding to the equivalent complexes of nNOS, while N(omega)-nitro-L-arginine (NNA) binding produces a state which appears intermediate in character between the nNOS NNA and arginine complexes. These spectroscopic studies have permitted the determination of ligand-specific high-spin states which reveal similarities and differences between iNOS and nNOS.

Selenite incubated with NADPH and mammalian thioredoxin reductase yields selenide, which inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron.

Selenite and selenodiglutathione (GS-Se-SG) efficiently inhibited 5-lipoxygenase activity in sonicates of human monoclonal B-lymphocytes. The apparent IC50 of GS-Se-SG was 0.5 microM. The inhibitory effect of these compounds was observed within 10 min of incubation. In order to elucidate if the mechanism of inhibition by these compounds was result of direct interference with lipoxygenase or indirectly mediated by cellular factors, pure 15-lipoxygenase from soybeans was used as a model system for enzyme assays and electron spin resonance (ESR) measurements. Incubation of 15-lipoxygenase with a mixture of human placenta thioredoxin reductase (TR) or calf-thymus TR, selenite, and NADPH blocked the activity of the enzyme. Neither TR and NADPH nor selenite inhibited soybean lipoxygenase when incubated separately. These results suggest that selenite must be reduced to selenide in order to inhibit 5- and 15-lipoxygenase activities. Preincubation anaerobically of 15-lipoxygenase with chemically generated selenide (6 microM) resulted in a strong inhibition of activity, in assays with arachidonic acid in the presence of oxygen. In contrast, selenide exposed to air prior to preincubation did not inhibit the enzyme. Since selenide is known to be efficiently oxidized by oxygen and to form elemental selenium the results evidence that selenide was the inhibitor of lipoxygenase activity in the anaerobic preincubations. After incubation with TR, NADPH, and selenite or with chemically generated selenide, the ESR spectrum of 15-lipoxygenase changed: the dominant axial component with a peak at g = 6.1 decreased, and a rhombic form with a feature at g = 4.28 grew. The results suggest that selenide produced by the reduction of selenite reduces the active site iron to the ESR invisible state and changes the ligation geometry of the oxidized form.

Organolanthanide tetrahydroborates. Ligation geometry and coordinative saturation

Organolanthanide tetrahydroborates. Ligation geometry and coordinative saturation