Spin-state Equilibrium Research Articles

Heterometal Clusters Containing the Cuboidal Fe4S3 Fragment: Synthesis, Electron Distribution, and Reactions

The heterometal clusters MFe4Ss(PEt3)&1 [M = V (l), Mo (2)] contain a cuboidal Fe& fragment that is bridged by three p2-S atoms to the heterometal, forming a MFe& core structure of idealized C3v symmetry. Three symmetry-related (equatorial) Fe atoms and the M atom have terminal phosphine ligands while the unique (axial) Fe atom is terminally coordinated by chloride. These clusters and others prepared in this work are of primary interest because their Fe4@3-S)z thiolate is bound at the axial site to form tetrahedral FeS3(SR) units. Molybdenum clusters 2 and 4 exhibit dimensionally expanded cuboidal fragments compared to vanadium clusters 1 and 3. Collective structural and Mossbauer and 'H NMR spectroscopic results indicate that the additional valence electron in the molybdenum clusters is largely delocalized in the Fe& cuboidal portion. Cluster 1 exhibits the f i s t example of a spin state equilibrium (S = 0 t S = 1) in any iron-sulfur cluster. Clusters 2 and 4 have S = l/2 or spin-admixed (S = l / ~ , 3/2) ground states. All clusters are readily identified by their characteristic lH NMR spectra, in which the axial thiolate substituents exhibit contact shifts indicative of negative spin delocalization.

Coordination and spin state equilibria as a function of pH, ionic strength, and protein concentration in oxidized dimeric Scapharca inaequivalvis hemoglobin.

The oxidized homodimeric Scapharca inaequivalvis hemoglobin undergoes changes in coordination and spin state as a function of pH, ionic strength, and protein concentration which have been monitored by optical absorption spectroscopy. Three species contribute to the spectra between pH 5.8 and 8.7: (i) a hexacoordinate high spin aquomet derivative, whose concentration is essentially constant over the whole pH range analyzed; (ii) a pentacoordinate high spin component which prevails at alkaline pH values, and (iii) a hexacoordinate low spin hemichrome, which is formed at acid pH. The contribution of each of the components to the observed spectra was calculated with the singular value decomposition procedure and has been described quantitatively in terms of a linkage scheme which accounts for the change in heme coordination and for the observation that the high spin to low spin transition entails dissociation into monomers. An important feature of the linkage scheme is the cooperative binding of protons to aquomet dimers. Stopped flow experiments to study the kinetics indicate that dissociation into monomers is the rate-limiting process. The unusually strong tendency of oxidized HbI to loose the heme-bound water molecule is discussed in terms of strain in the iron-proximal histidine bond.

Resonance Raman spectroscopic studies of ligated mutant myoglobins

Ligand binding (CO and N 3 −) to wild-type porcine myoglobin and to several mutant forms, expressed and purified from E. coli cells, has been studied using Raman spectroscopy. The ν(FeCO) stretching vibration in Mb IICO has been compared for the wild-type and mutant proteins. This gives a broad band consisting of five components, indicating five possible configurations of the bound CO. The distal pocket mutants show large variations in bandshape, the major component occurring at progressively lower wavenumber in the order: wild-type (WT) > E11 Val → Thr (VT) > E7 His → Val (HV) > the double mutant VT HV (M2) . Changes observed in the Raman band assigned to the azide bending mode in Mb IIIN 3 have been interpreted in terms of resonance structures involving two forms of azide binding. Repulsion between the bound azide ligand and the OH group of the adjacent thr residue in the VT mutant, and a shorter FeN(his) bond in the proximal mutant Ser → Leu (F7), both affect this bonding. In the wild-type protein (WT), hydroxymetmyoglobin exists in a spin-state equilibrium which, at room temperature, is predominantly high-spin. In the F7 mutant this equilibrium is shifted in favour of the low-spin form. A low-spin iron species also exists in the aquometmyoglobin form of this mutant.

Spin-state equilibria in non-aqueous solution and quantum-mechanical investigations of iron(II) and nickel(II) complexes with 4-substituted 2,6-bis(benzimidazol-2-yl)pyridines

Cationic complexes with a series of tridentate ligands, L = 4X-substituted 2,6-bis(benzimidazol-2-yl)pyridines, [ML2][ClO4]2(M = Fe or Ni; X = H, OH or Cl), were isolated and characterized, together with the free pyridines, by elemental analysis, Fourier-transform IR, 1H NMR and UV/VIS spectroscopy. The syntheses were performed via condensation of o-phenylenediamine with 4-substituted pyridine-2,6-dicarboxylic acids. Ligand-field parameters were estimated for the nickel complexes. The [FeL2]2+ species show thermally induced spin-crossover behaviour (1A1→5T2g) which has been investigated in methanol, nitromethane and 20%(v/v) dimethylformamide in MeOH. The behaviour is complicated by two complex dissociation equilibria, for which equilibrium constants have been evaluated. Ligand substitution is reflected in a change of the spin state in solution (µexptl= 2.50, X = H; 4.19, OH; and 4.49 µB, Cl at 295 K, in MeOH) and in the metal-to-ligand charge-transfer band (500–557 nm); when M = Fe and X = H there is a pronounced spin-crossover equilibrium in methanolic solution (µexptl= 1.31–3.45 µB for 213–328 K). A small variation of the magnetic moments when M = Fe and X = OH (µexptl= 3.77–4.73 µB at 220–332 K) might indicate a temperature-variable population of the 5Eg sublevel or variation in hydrogen bonding. The results are compared with quasi-relativistic quantum-mechanical calculations, and the spin-crossover behaviour of the new ligands, L, with substituents X = CHO, NH2, CN, Me, NO2, OH, CONH2, COCl, SH, F, Cl, Br or I has been estimated. The differences in the calculated heats of formation between the high-and low-spin forms of [FeL2]2+ when plotted against Δδ(=1H NMR para increment for substituents X in benzene) show a turning point in the region around X = H and in this region spin-crossover behaviour is observed. Outside this region there is very little or no such behaviour and it is therefore possible to predict the spin-crossover behaviour for other substituents X from the Δδ value.

Complexes of functionalised phosphine ligands. Part 1. Complexes of FeIII, CoIII, NiIIand ReVwith tridentate Schiff bases having PNO, NNO and NNS donor sets. Crystal structures of 2-(Ph2PC6H4NCH)C6H4OH and [Co{2-(Ph2PC6H4CHN)C6H4O}2][PF6

The Schiff bases 2-[Ph2P(CH2)nNCH]C6H4OH (n= 3, HL1 or 2 HL2), 2-(RCHN)C6H3(OH)X-4 (R = 2-Ph2PC6H4, X = H HL3; R = 2-C5H4N, X = H HL4; R = 2-C5H4N, X = Cl HL5) were synthesised from the appropriate amine and aldehyde. On deprotonation these all functioned as tridentate monoanionic ligands to give complexes [FeL2]+ and [CoL2]+ with FeIII and CoIII and neutral complexes of stoichiometry NiL2 with NiII. The iron complexes were examined by Mossbauer spectroscopy which indicated the presence of two iron sites in [FeL12]+ with a spin-state equilibrium dependent on both temperature and the counter ion. The complex [FeL32]+ showed a single iron site, again with a spin state dependent on counter ion and temperature. The crystal structures of HL3 and [CoL32]+ have been determined. The distortions in free HL3 predispose it for co-ordination in a fac geometry to the Co with cis-PPh2 groups, and the changes occurring on co-ordination are discussed in detail. Reaction of RCHO (R = 2-Ph2PC6H4 or 2-C5H4N) with 2-aminobenzenethiol gave stable thiazoles R[graphic omitted]-2 which did not ring open to give tridentate ligands even on reaction with base and/or metal ions.

Reaction of arylcarbenes with methanol: triplet-state reactivity or spin-state equilibrium as a moving target?

Reaction of arylcarbenes with methanol: triplet-state reactivity or spin-state equilibrium as a moving target?

Solvent effects on the singlet - triplet equilibrium and reactivity of a ground triplet state arylalkyl carbene

Results from intramolecular singlet and triplet specific reactivity in solvents of different Polarity suggest that the spin state equilibrium of 1,2-diphenyl-1-butylidene, a triplet ground state carbene. is largely susceptible to solvent polarity. The results are consistent with stabilization of the zwitterionic singlet state in solvents of high polarity.

Cytochrome P450-benzphetamine interactions in the endoplasmic reticulum: studies using a monoclonal antibody to P450b.

A monoclonal antibody (MAb) to phenobarbital-induced rat cytochrome P450b was used to study the interaction of the substrate benzphetamine (Bz) with cytochromes P450 in liver microsomes. Binding of Bz to liver microsomes from phenobarbital-treated rats was monitored by the substrate-induced type I spectral change. The MAb maximally inhibited this spectral change by 49%, providing a probe to distinguish MAb-specific P450b from other Bz-binding P450s. Thermodynamic parameters of the interaction were determined in the absence and presence of MAb. The MAb did not influence the spin-state equilibrium of substrate-free P450b, but it increased the low spin content of substrate-bound P450b. The MAb also decreased the affinity of both high and low spin P450b for Bz. The temperature dependence of the Bz-binding interactions revealed a transition near 20 degrees C. Fluorescence polarization measurements of the membrane probe 1,6-diphenyl-1,3,5-hexatriene also revealed a transition at this temperature. The MAb comparably inhibited Bz binding to high spin P450b in the low and high temperature regions, whereas MAb inhibition of Bz binding to low spin P450b was greater in the low temperature region than in the high temperature region. These results indicate temperature-dependent changes in membrane structure that modulate both Bz binding to P450b and MAb-P450b-Bz interactions. These results also demonstrate the utility of MAbs for evaluating P450-substrate binding microequilibria of MAb-specific P450s in the presence of other P450s while in the natural membrane environment of the endoplasmic reticulum.

The cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. 1. Thermodynamic analysis.

We have investigated the spin-state equilibrium of adrenal mitochondrial P450scc (cholesterol-side-chain-cleaving, CYP11A1) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high-spin/low-spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non-linearity resolves clearly into two phases when the temperature range is extended from 37 degrees C to -20 degrees C using ethylene glycol as anti-freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are delta HA = 0.7 kJ.mol-1 and delta SA = 5J.K-1.mol-1 at low temperatures and delta HB = -42 kJ.mol-1 and delta SB = -152 J.K-1.mol-1 at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH. The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 +/- 0.5 higher in the low-temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.

The cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. 2. Conformational analysis.

We have confirmed and characterised structurally the enzyme conformational changes deduced from the preceding thermodynamic analysis of the adrenal mitochondrial cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. The spin-transition kinetics following rapid pH jumps were multiphasic in aqueous buffer and biphasic in the presence of 35% ethylene glycol. The activation energy between -2 degrees C and 30 degrees C of both phases was exceptionally high (Ea = 147 kJ.mol-1), suggesting the involvement of large-scale conformational changes. The pH and temperature effects on the CD spectrum show that the enzyme is in equilibrium between at least two conformations which are predicted by the thermodynamic model, but which are not directly correlated to the spin state. The CD changes between 260 nm and 280 nm indicate that the conformation prevailing at high temperatures is characterised by a decreased polarity of the tyrosine environments; the changes between 200 nm and 250 nm suggest furthermore a 4% decreased protein helical content.

Substrate mobility in a deeply buried active site: analysis of norcamphor bound to cytochrome P-450cam as determined by a 201-psec molecular dynamics simulation.

While cytochrome P-450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) stimulation of norcamphorbound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the heme iron and the substrate of about 1.0 A; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphorbound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex.

Pressure-induced spin-state isomerizations in weakly tetragonal Nickel(II) complexes with diethylthiourea

The ability of high pressure to influence spin-state equilibria in (Ni(detu){sub 4}X{sub 2}) (detu = N,N{prime}-diethylthiourea; X = Cl, Br, I) is demonstrated. The complexes are six-coordinate and weakly tetragonal. An equilibrium between singlet and triplet isomers is present. High-pressure shifts the equilibrium toward the triplet isomer in each complex. The equilibria are monitored spectroscopically. The results represent the first examples of low-spin to high-spin conversion via pressure-induced changes in molecular geometry.

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding.

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.

Some properties of an inhibitory cytochrome P-450 metabolic-intermediate complex existing in a spin-state equilibrium

Incubation, in the presence of NADPH/O 2, of the type II compound revenast with a partially solubilized, phenobarbital-induced rat liver microsomal cytochrome P-450 system results in the formation of a difference spectrum exhibiting a Soret band at 407 nm and a trough at 423 nm. Experiments with N 2 and metyrapone suggest this spectral perturbation to originate from binding to the hemoprotein of a metabolic intermediate derived from the amine substrate. The percentage of pigment complexed can be assessed to be about 7%, with half-maximal complexation occurring at a revenast concentration of 125 μM. Adduct formation is inhibitory to the N-hydroxylation of 4-chloroaniline and N-demethylation of N, N-dimethylaniline; kinetic analysis suggests a competitive interaction at the catalytic site of the reactive revenast derivative with the tertiary arylamine. Inhibition of the two monooxygenation reactions is reversible, indicating weak heme bonding of the active intermediate. This behaviour differs from that of most other product adducts so far examined. The aberrant functional properties of the metabolic adduct appear to reflect complex molecular organization of this compound, as is also evidenced by the unique position of the Soret region absorption maximum at 407 nm; this spectral data hints at the presence of a mixture of high- and low-spin forms. Indeed, chemical reduction or oxidation of the product complex reveals the existence of a low-spin component hidden in the metabolic spectrum. Model studies suggest that the product adduct possibly arises from N-oxidation of the revenast molecule.

Spin equilibria in human methemoglobin: effects of bezafibrate and inositol hexaphosphate as measured by susceptometry and visible spectroscopy

The effects of inositol hexaphosphate (IHP) and a second allosteric effector, bezafibrate, on the spin-state equilibria of the mixed-spin derivatives of ferric human hemoglobin A are examined. Changes in spin-state equilibrium are monitored by measuring absorption spectra in the visible region (460-700 nm) as well as by direct measurements of magnetic susceptibility by means of a superconducting fluxmeter. The addition of IHP at pH 6.5 results in a measurable shift in the spin equilibria of these derivatives toward higher spin. However, the addition of bezafibrate in the presence of IHP results in still larger shifts toward the high-spin form. The changes in the free energies of the spin-state equilibria resulting from the combination of these two effectors are similar in magnitude to that which results from the R-state to T-state transition in carp hemoglobin.

Reactivity ofN-trimethylsilyl-imidazole and 2-methylimidazole; synthesis and physico-chemical studies on transition metal imidazolates and mixed ligand imidazolates

N-trimethylsilylimidazole andN-trimethylsilyl-2-methylimidazole react with MCl2 yield [ML2] (M=Ni or Cu; L =imidazolate or 2-methylimidazolate). The complex [NiL2] can also be prepared from [Ni(Ph3P)2Cl2]; [Cu(Ph3P)3Cl] yields [Cu(Ph3P)2L]. The complexes are soluble in most non-polar organic solvents and experimentally determined molecular weights suggest they are monomers in solution. Magnetic susceptibility measurements and reflectance and electronic spectral studies at room temperature are commensurate with square-planar geometry for [CuL2]. Although, the ligand field spectrum for [NiL2] is compatible with a square-planar geometry, the anomalous μeff value suggests that the ground electronic state of NiII in [NiL2] is near the magnetic cross-over point concomitant with the singlet-triplet spin state equilibrium. The i.r. spectra exhibit characteristic imidazolate and Ph3P ring vibrations, andv(M−N) andv(M−P) stretching vibrations at the appropriate frequencies. The synthesis and characterization of a CuII complex, formulated as [Cu(Ph3P)2LCl], with tetrahedral structure is described.

Studies on the linkage between spin equilibria and protein structure in carp ferric hemoglobin

The effects of protein conformation on the spin-state equilibria of several derivatives of carp hemoglobin have been examined. This has been done by measuring the pH dependence of the paramagnetic susceptibilities of these derivatives in the presence and absence of inositol hexakisphosphate, P6-inositol. In all cases the addition of P6-inositol at low pH and the lowering of the pH in the presence of P6-inositol shift the spin-state equilibrium in favor of the high-spin electronic configuration. The P6-inositol and pH dependence of these magnetic properties parallels the pH and P6-inositol dependence of the conformational state of the hemoglobin as determined in earlier studies and further supports a thermodynamic linkage between the electronic state of the iron atoms and the quaternary structure of the hemoglobin molecule.

Temperature jump relaxation kinetics of the P-450cam spin equilibrium.

The ferric spin-state equilibrium and relaxation rate of cytochrome P-450 has been examined with temperature jump spectroscopy using a number of camphor analogues known to induce different mixed spin states in the substrate-bound complexes [Gould, P., Gelb, M., & Sligar, S. G. (1981) J. Biol. Chem. 256, 6686]. All temperature-induced spectral changes were monophasic, and the spin-state relaxation rate reached a limiting value at high substrate concentrations. The ferric spin equilibrium constant, Kspin, is defined in terms of the rate constants k1 and k-1 via Kspin = k1/k-1 = [P-450(HS)]/[P-450(LS)] where HS and LS represent high-spin (S = 5/2) and low-spin (S = 1/2) ferric iron, respectively, and the spectrally observed spin-state relaxation rate by kobsd = k1 + k-1. A strong correlation between the fraction of high-spin species and the rate constant, k-1, is observed. For a 3 degrees C temperature jump (from 10 to 13 degrees C), the 23% high-spin tetramethylcyclohexanone complex (Kd = 45 +/- 20 microM) is characterized by a ferric spin relaxation rate of kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and camphoroquinone (75% high spin, Kd = 15 +/- 5 microM) complexes are 1430 and 346 s-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)