The effect of temperature on the spectroscopic properties of the heme c octapeptide from cytochrome c.

The effect of temperature on the optical properties of the acetylated heme c octapeptide from cytochrome c was examined. At above ambient temperatures the observed optical spectrum with maxima at 549 and 424 nm was characteristic of high-spin ferrous hemeproteins. At below ambient temperatures the optical spectrum became characteristic of low-spin ferrous hemeproteins with maxima at 547, 518, and 410 nm. A thermodynamic characterization of this two component system yielded a deltaHO of -10.1 +/- 0.7 kcal/mol and a delta S0 of-37.6 +/- 2.5 e.u. for the temperature dependent process. Discussion of the spectroscopic and thermodynamic parameters was presented in terms of the consistent magnetic and structural properties of heme complexes.     

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.     

Spectral Studies of Iron Coordination in Hemeprotein Complexes: Difference Spectroscopy below 250 mμ

In order to evaluate the feasibility of observing the spectral behavior of protein groups in the coordination sphere of the iron in hemeproteins, criteria are developed to determine whether or not the application of difference absorption spectroscopy to the study of complex formation will be successful. Absolute absorption spectra, 300-1100 mmu, from bacterial catalase complexes are displayed, and the infrared bands correlated with magnetic susceptibility values of similar complexes of other hemeproteins. Dissociation constants for the formation of cyanide and azide complexes of metmyoglobin, methemoglobin, bacterial catalase, and horseradish peroxidase are given. Difference spectra, 210-280 mmu, are displayed for cyanide and azide complexes of these hemeproteins. A band at 235-241 mmu is found in the difference spectra of all low-spin vs. high-spin complexes. The factors which favor the assignment of this band to a transition involving a histidine residue are presented.     

Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.     

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.

The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.     

Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.     

Some iron(III) complexes with polydentate Schiff base ligands

The iron(III) complexes of three Schiff base ligands are studied as their chloride or perchlorate salts and their electronic spectra, EPR spectra, and electrochemical behavior reported. Two of these ligands are formed from reaction between salicylaldehyde and 9 or 12-membered tri- or tetraazalkanes. EPR evidence indicates that one of the complexes, [1,12-bis(2-hydroxybenzylidene)-(1,4,9,12-tetraazadodec- 6-ene)iron(III)]perchlorate-1,5-water, is a spin-crossover species containing both high-spin and low-spin iron(III) in equilibrium. The third ligand comes from pyrrole-2-carboxaldehyde and a tetraazadodecane.     

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH₂-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H₂O₂ without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O₂) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.     

1H NMR studies of heme pocket conformation in zinc-substituted leghemoglobin, a diamagnetic analog of deoxyleghemoglobin

Reconstitution of apoleghemoglobin with zinc protoporphyrin IX is reported. NMR spectra show that the reconstitution is orientation specific and that there is no detectable heme isomerism or conformational heterogeneity. Resonances of heme substituents and distal and proximal amino acid protons have been assigned. Only minor differences in porphyrin-protein packing occur between zinc leghemoglobin and the CO complex of ferrous leghemoglobin. The zinc is five-coordinate and is ligated by the proximal histidine. Comparisons with diamagnetic six-coordinate complexes show that the distal His-61 and Leu-65 side chains move away from the binding site upon coordination of exogenous ligands. Conformational changes are minimal when the ligand is O2.     

Resonance Raman studies of lactoperoxidase

Resonance Raman (RR) spectra obtained at three excitation wavelengths are reported for various ferric, ferrous, and ferryl derivatives of bovine lactoperoxidase. The RR spectra of the ferric derivatives show the full complement of the vinyl stretching and scissor modes indicating that the two vinyls in the protoporphyrin IX prosthetic group are present in unmodified forms. The cysteine thiol complex exhibits a RR spectrum identical to that of the native enzyme, an observation which strongly suggests a nonheme binding site for the thiol substrates. The different ferrous complexes of lactoperoxidase which result from heme reduction at slightly alkaline and acidic pH gave identical low-frequency RR spectra. Differences are observed, however, in the high-frequency region. Reduction in the presence of cyanide, however, yields two time-resolved complexes. Changes in the ligand field during the conversion to the final form of the cyanoferrous complex are proposed based on the changes observed in the low-frequency vibrational spectrum. Comparisons are made between the low-frequency RR spectra of the limiting form of the cyanoferrous and the nitric oxide lactoperoxidase complexes. The similarity between the RR spectra of these two complexes in the 150-500 cm-1 region supports the assignment of structures for these complexes where the six-coordinate heme iron is displaced from the heme plane and away from the proximal histidine ligand.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

The diverse spectroscopic properties of ferrous cytochrome P-450-CAM ligand complexes

The UV-visible absorption, magnetic circular dichroism (MCD) and CD spectral characteristics of a variety of low spin ferrous P-450-ligand complexes have been carefully determined in order to establish whether all such complexes are hyperporphyrins as previously suggested in the literature. Two general spectral classes are found to occur. Complexes in the first class are, indeed, hyperporphyrin in nature, with pi-acceptor ligands such as CO, NO, phosphine, nitrosoalkanes and isocyanides trans to cysteinate. Individual, but minor, variations in the spectral properties of the hyperporphyrins suggest that subclasses exist, wherein the nature of the trans ligand to thiolate affects the orbital overlap pattern and thus the observed spectra. Adducts in the second spectral class, which have sigma-donor nitrogen and sulfur ligands, also have the red-shifted Soret absorption maximum but are spectrally distinct in all other respects from the hyperporphyrins. Comparison of the MCD spectra of the second category to those of ferrous cytochromes b5, c, and P-420 suggests that the axial cysteinate ligand is still present in the nonhyper ferrous P-450 species. Thus, the combination of a strongly electron-donating cysteinate ligand and a trans sigma-donor, not the orbital mixing mechanism, is most likely the origin of the red-shifted Soret absorption maximum of nonhyper ferrous P-450 ligand complexes. Further, the nature of the total electronic interactions between both axial ligands and the heme iron of ferrous P-450 and not solely the cysteinate ligand determines whether the ligand complexes will be of the hyper or nonhyperporphyrin category. These findings are strengthened by the simultaneous use of three different spectroscopic techniques; together they provide a more detailed explanation for the unusual spectroscopic properties of cytochrome P-450.     

Solvent effect on MCD of Fe(III) heme complexes: magnetic circular dichroism spectra of five-coordinated high-spin iron(III) protoporphyrin-IX-dimethylester in the visible region and their environmental effect. A characterization of the visible electronic transitions in Fe(III) high-spin porphyrins

Magnetic circular dichroism (MCD) spectra were observed to characterize the nature of the visible bands for high-spin Fe(III) protoheme derivatives with p-nitrothiophenolate, p-nitrophenolate, and methoxy anion as the fifth ligands in several solvents. The visible MCD bands for p-nitrophenolate heme were very sensitive to the solvent polarity, but that those for p-nitrothiophenolate heme and methoxy heme were not dependent on solvent polarity. Thus, both the two visible MCD band positions and the magnitudes were dependent on ET value (a solvent polarity parameter) in the former complex, but not in the latter two complexes. The results are consistent with previously proposed electronic structures of high-spin Fe(III) heme complexes.     

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.     

Pentacoordinate hemin derivatives in sodium dodecyl sulfate micelles: model systems for the assignment of the fifth ligand in ferric heme proteins.

Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.     

Models of the two heme centers in cytochrome oxidase. The optical properties of cytochrome a and a3

High and low spin complexes of ferric and ferrous heme a have been prepared and characterized spectroscopically. Bis(1-methylimidazole) heme a provides a good model for cytochrome a in both oxidation states while several spectral properties of cytochrome a3 can be reproduced by 1,2-dimethylimidazole heme a3. The visible absorbance spectra of these analogs account well for the absorbance spectra of oxidized and reduced cytochrome oxidase and support the conclusion (Vanneste, W. (1966) Biochemistry 5, 838-848) that cytochrome a provides the major contribution to the spectral changes in the 600 nm band upon reduction. The 655 nm band present in cytochrome oxidase appears to be a characteristic of high spin heme a+3.     

1H nmr studies of complexes of ferric leghemoglobin with substituted pyridines and nicotinic acids

The ¹H nuclear magnetic resonance (nmr) spectra of complexes of soybean ferric leghemoglobin with 3-substituted pyridines and 5-substituted nicotinic acids have been recorded in order to determine the influence of axial ligands on heme electronic structure. The hyperfine shifted resonances of the heme group were assigned by analogy to previous assignments for the pyridine and nicotinic acid complexes of leghemoglobin. The spectra are characteristic of predominantly low-spin ferric heme complexes. For the pyridine complexes, the rate of ligand exchange was found to increase with decreasing ligand pK_A. For many of the complexes, optical and nmr spectra reveal the presence of an equilibrium mixture of high- and low-spin states of the iron atom. The percentage of high-spin component increases with decreasing ligand pK_A Smaller hyperfine shifts are noted for leghemoglobin complexes with ligands capable of weak ligand → metal π bonding. The pattern of hyperfine shifted resonances is similar for all complexes studied and indicates that the overall heme electronic structure is dominated by the bonding to the proximal histidine.     

Complexes between synthetic polymer ligands and ferri-delta and ferro-protoporphyrin IX.

The complexes of synthetic polymer ligands, i.e. poly-L-lysine, poly-4-vinyl-pyridine, poly-N-vinyl-2-methylimidazole and the higher branched polyethyleneimine, with ferri- or ferro-protoporphyrin IX were studied from the standpoint of polymer ligand effects by comparison with those of their monomeric model ligand complexes and poly-gamma-benzyl-L-glutamate containing an imidazole nucleus at the chain end. The coordination numbers and formation constants were determined optically and their structures were also estimated. The coordination number of a poly-L-lysine complex was two, but those of other polymer ligand complexes were one. One of the polymer effects, which was indicated by the large formation constants of the polymer complexes, was caused by the increment of the local ligand concentration around the polymer chain. Another was caused by the conformational effect of an alpha-helical structure in the poly-L-lysine complexes. The interaction of a poly-L-lysine-heme complex with molecular oxygen was also studied. An observed pseudo-allosteric phenomenon may be due to the specific structure of a poly-L-lysine complex which is different from those of other polymer ligand complexes.     

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.     

Substitution of trans ligands in μ-oxo-bis(μ-acetato)diruthenium(III) complexes: Synthesis and kinetic studies

[Ru₂O(L)₆(acetate)₂](PF₆)₂ {L = pyridine 1; 4-picoline 2} undergo aquation in acetone-water (60:40 v/v) mixed solvent to form diaquo complexes in solution as shown by proton NMR studies. Ligands trans to the μ-oxo group are substituted. These diaquo complexes react with substituted pyridines and imidazoles to form respective disubstituted complexes. Rate constants for aquation and complexation under pseudo first order conditions of ligand are reported. Rate constants increase with increase in the basicity of incoming ligand. Disubstituted complexes proposed to be formed in solution have been isolated and characterized by elemental analyses, visible spectra, proton NMR. Single crystal X-ray structures of 4-picoline and 4-methylimidazole disubstituted complexes are reported. All the isolated complexes exhibit a strong peak between 570 and 585 nm in their visible absorption spectra. λ_max varies linearly with ∑pk_a of terminal ligands. In disubstituted complexes of 1 with 2-methyl and 4-methyl imidazole deprotonation of N(1)H of methylimidazoles takes place in solution.