Redox-dependent structural changes in an engineered heme-copper center in myoglobin: insights into chloride binding to CuB in heme copper oxidases

The effects of chloride on the redox properties of an engineered binuclear heme-copper center in myoglobin (Cu(B)Mb) were studied by UV-vis spectroelectrochemistry and EPR spectroscopy. A low-spin heme Fe(III)-Cu(I) intermediate was observed during the redox titration of Cu(B)Mb only in the presence of both Cu(II) and chloride. Upon the first electron transfer to the Cu(B) center, one of the His ligands of Cu(B) center dissociates and coordinates to the heme iron, forming a six-coordinate low-spin ferric heme center and a reduced Cu(B) center. The second electron transfer reduces the ferric heme and causes the release of the coordinated His ligand. Thus, the fully reduced state of the heme-copper center contains a five-coordinate ferrous heme and a reduced Cu(B) center, ready for O(2) binding and reduction to water to occur. In the absence of a chloride ion, formation of the low-spin heme species was not observed. These redox reactions are completely reversible. These results indicate that binding of chloride to the Cu(B) center can induce redox-dependent structural changes, and the bound chloride and hydroxide in the heme-copper center may play different roles in the redox-linked enzymatic reactions of heme-copper oxidases, probably because of their different binding affinity to the copper center and the relatively high concentration of chloride under physiological conditions.     

A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.

Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.     

Thermodynamic redox behavior of the heme centers in A-type heme-copper oxygen reductases: comparison between the two subfamilies

The study of the thermodynamic redox behavior of the hemes from two members of the A family of heme-copper oxygen reductases, Paracoccus denitrificans aa₃ (A1 subfamily) and Rhodothermus marinus caa₃ (A2 subfamily) enzymes, is presented. At different pH values, midpoint reduction potentials and interaction potentials were obtained in the framework of a pairwise model for two interacting redox centers. In both enzymes, the hemes have different reduction potentials. For the A1-type enzyme, it was shown that heme a has a pH-dependent midpoint reduction potential, whereas that of heme a₃ is pH independent. For the A2-type enzyme the opposite was observed. The midpoint reduction potential of heme c from subunit II of the caa₃ enzyme was determined by fitting the data with a single-electron Nernst curve, and it was shown to be pH dependent. The results presented here for these A-type enzymes are compared with those previously obtained for representative members of the B and C families.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Metal-binding properties of an engineered purple CuA center in azurin

A CUA center engineered into Pseudomonas aeruginosa azurin was studied by metal substitution. Metal-binding properties were determined by electronic absorption (UV-vis) and electrospray ionization mass spectrometry (ESI-MS). The metal-binding site readily binds thiophilic metal ions, such as Hg(II), Ag(I), Cu(I), Cd(II), and Au(I). Harder metal ions, like Co(II), bind to apo-CuA-azurin only under basic conditions (pH 9.1-9.2). The results obtained from these studies indicate that two factors influence metal binding in CuA azurin: (1) the site favors metal combinations which produce an overall +3 charge, and (2) the site binds soft, thiophilic metal ions. The results demonstrate the remarkable ability of the CuA center to maintain valence delocalization of its native metal ions and to ensure redox accessibility of only one of the two redox couples (i.e., [Cu(1.5)...Cu(1.5)]<==> [Cu(I)...Cu(I)]) under physiological conditions. These findings may lead to the preparation of new metal ion derivatives and can serve as a basis for understanding this efficient electron transfer center.     

Structural and dynamic properties of the heme pocket in myoglobin probed by optical spectroscopy

We report the optical absorption spectra of sperm whale deoxy-, oxy-, and carbonmonoxymyoglobin in the temperature range 300–20 K and in 65% glycerol or ethylene glycol–water mixtures. By lowering the temperature, all bands exhibit half-width narrowing and peak frequency shift; moreover, the near-ir bands of deoxymyoglobin show a marked increase of the integrated intensities. Opposed to what has already been reported for human hemoglobin, the temperature dependence of the first moment of the investigated bands does not follow the behavior predicted by the harmonic Franck–Condon approximation and is sizably affected by the solvent composition; this solvent effect is larger in liganded than in nonliganded myoglobin. However, for all the observed bands the behavior of the second moment can be quite well rationalized in terms of the harmonic Franck–Condon approximation and is not dependent on solvent composition. On the basis of these data we put forward some suggestions concerning the structural and dynamic properties of the heme pocket in myoglobin and their dependence upon solvent composition. We also discuss the different behaviors of myoglobin and hemoglobin in terms of the different heme pocket structures and deformabilities of the two proteins.     

Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin.

Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.     

Modification of the heme distal side in myoglobin by cyanogen bromide. Heme environmental structures and ligand binding properties of the modified myoglobin

Met, deoxy, and CO forms of myoglobin (Mb) react with a stoichiometric amount of cyanogen bromide (BrCN) to cause substantial changes in the 1H NMR, optical absorption, and infrared spectra. These spectral changes were interpreted as arising from the substantial alterations in the heme environments, most probably due to the modification of the histidine residue at the heme distal side. It is also revealed that the modified Mb does not combine with some exogenous ligands such as CN-, CH3NH2, and O2, although it does with N-3 or CO. These unique ligand binding properties are also discussed with relevance to a role of the distal histidine in stabilizing the coordinated ligand through a hydrogen bond and to a steric constraint.     

Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin.

Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.     

Physical-chemical studies on the role of the metal ions in concanavalin A.

Substitution of Co²⁺ for Mn²⁺ in concanavalin A generates characteristic circular dichroism and magnetic circular dichroism spectra which are strongly affected by the concentration of Ca²⁺. With three equivalents of Ca²⁺ per protomer of [(Co²⁺)Con A], no spectral effects of addition of α-methyl-d-glucopyranoside can be demonstrated. With one equivalent of Ca²⁺, however, α-methyl-d-glucopyranoside alters the circular dichroism and magnetic circular dichroism spectra in a manner identical to that produced by adding further equivalents of Ca²⁺. Under these same conditions the higher molecular weight carbohydrates, trehalose and melezitose, cause no spectral alterations in the regions investigated.The magnetic circular dichroism spectrum of [(Co²⁺)Con A] is characterized by a negative peak centered at 510 nm (θ/gauss = ?0.28 °) and a pronounced shoulder at 462 nm (θ/gauss = ? 0.16 °). Comparison of this spectrum to that of Co(H₂O)₆²⁺ indicates that the transition metal ion exhibits octahedral geometry in solution and maintains this geometry in its interaction with carbohydrate moieties.Circular dichroism experiments in the far ultraviolet region indicate a change in secondary (presumed β) structure upon interaction of Apo Con A with Mn²⁺ consistent with a more ordered arrangement. Unlike Mn²⁺, cobalt alone will not induce these secondary changes until Ca²⁺ is added. Kinetic analysis, using a mannan light scattering assay, indicates that [(Mn²⁺)Con A] and [(Co²⁺)Con A] will slowly recover cross-linking function in the absence of Ca²⁺, suggesting that the role of the metal in S₂ is to accelerate a conformational change leading to binding or effector function.Overall, the data are consistent with a suggestion by Cuatrecasas (1973) that α-methyl-d-glucopyranoside binds to a locus different from the membrane binding (or agglutination) site. Nevertheless, there are strong conformational interactions between these two sites, since α-methyl-d-glucopyranoside will elute Con A from membrane surfaces.     

Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K_M and V_max values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC₅₀ values, K_i constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag⁺; Cd²⁺, Cr²⁺ and Mg²⁺.     

Azospirillum brasilense glutamine synthetase: influence of the activating metal ions on the enzyme properties

The kinetic properties of the Mg²⁺-activated and Mn²⁺-activated glutamine synthetase (GS) of Azospirillum brasilense in the biosynthetic reaction were studied. The Mg²⁺-supported and Mn²⁺-supported GSs in an average state of adenylylation varied in pH optimum, maximum activity, saturation functions for ammonium and glutamate, affinity to substrates, and in the Me²⁺-ATP ratio required for the optimal enzyme activity. Seventeen other cations were tested for the maintenance of GS activity. The level of the latter and the kinetic behavior of the GS in A.brasilense is suggested to depend essentially on the concentrations of Mg²⁺, Mn²⁺ and Co²⁺, as well as on their ratio     

Axial ligation of the high-potential heme center in an Arabidopsis cytochrome b561

Arabidopsis has four putative, di-heme cytochrome b561 proteins, including one localized in the tonoplast (TCytb). From a comparative electron paramagnetic resonance (EPR), UV–Vis absorption and resonance Raman study, on wild type, H83A/H156A-TCytb and H83L/H156L-TCytb double mutants, it follows that the H83 and H156 residues are binding one of the two hemes. These measurements show that the high-potential heme site is situated at the cytoplasmic side of the membrane and allow the unambiguous differentiation between two models on the heme localization in cytochrome b561 proteins.     

Oxygen binding and redox properties of the heme in soluble guanylate cyclase: implications for the mechanism of ligand discrimination

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O(2), thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O(2) has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe(2+)-O(2)) when frozen at 77 K in the presence of O(2). The ligation of O(2) was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at -7 °C, with the extremely low affinity for O(2). The low O(2) affinity was not caused by a distal steric protein effect and by rupture of the Fe(2+)-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe(2+)-proximal His bond. Based on our results, we propose that the weak Fe(2+)-proximal His bond is a key determinant for the low O(2) affinity of the heme moiety of soluble guanylate cyclase.     

Probing the local electronic and geometric properties of the heme iron center in a H-NOX domain

Heme-Nitric oxide and/or OXygen binding (H-NOX) proteins are a family of diatomic gas binding hemoproteins that have attracted intense research interest. Here we employ X-ray absorption near-edge structure (XANES) spectroscopy to study the nitric oxide (NO) binding site of H-NOX. This is the first time this technique has been utilized to examine the NO/H-NOX signaling pathway. XANES spectra of wildtype and a point mutant (proline 115 to alanine, P115A) of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) were obtained and analyzed for ferrous and ferric complexes of the protein. This work provides specific structural characterization of the solution state of several Tt H-NOX ferrous complexes (− unligated, − NO, and − CO) that were previously unavailable. Our iron K-edges indicate effective charge on the iron center in the various complexes and report on the electronic environment of heme iron. We analyzed the ligand field indicator ratio (LFIR), which is extracted from XANES spectra, for each complex, providing an understanding of ligand field strength, spin state of the central iron, movement of the iron atom upon ligation, and ligand binding properties. In particular, our LFIRs indicate that the heme iron is dramatically displaced towards the distal pocket during ligand binding. Based on these results, we propose that iron displacement towards the distal heme pocket is an essential step in signal initiation in H-NOX proteins. This provides a mechanistic link between ligand binding and the changes in heme and protein conformation that have been observed for H-NOX family members during signaling.     

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg(2+), Ca(2+), Sr(2+) and Ba(2+) were used for clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg(2+), Ca(2+) and Sr(2+) are counterions. When Ba(2+) is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B-A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr(2+) induces the greatest structural transition.     

An adaptable spectroelectrochemical titrator: the midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase

Elaborations to an earlier design of an electron paramagnetic resonance (EPR) spectroelectrochemical titrator are described. While maintaining the anaerobic capabilities of the original design, a number of modifications and revisions have been introduced. The most significant modification is the use of a detachable spectral cell, making the apparatus modular and adaptable for multiple forms of spectroscopy. Additional modifications include removable reference, auxiliary, and working electrodes; modifications to facilitate sample transfer; and adaptations for operation within an anaerobic chamber. This apparatus has been used successfully in the coulometric titration of a [4Fe-4S] enzyme, as measured by EPR spectroscopy. The midpoint reduction potential for the 2+/1+ couple in the [4Fe-4S] cluster of lysine 2,3-aminomutase is -479+/-5mV, a value that falls within the range typical of ferredoxin-like iron-sulfur clusters.