Electronic Absorption Data Research Articles

Low band gap-conjugated polymer derivatives

Three new poly(4,4-dialkylcyclopentadithiophene (PDOCPT) derivatives, poly(4,4-dialkylcyclopentadithiophene-co-pyridine) (PDOCPTPy), poly(4,4-dialkylcyclopentadithiophene-co-phenanthroline) (PDOCPTPh), and poly(cyclopenta[2,1- b:3,4- b′]dithiophen-4-one) (PDOCPTCK) were prepared. They were synthesized form the Stille coupling of 2,7-bis-trimethyltin-4,4-dialkylcyclopentadithiophene with dibromopyridine and dibromophenanthroline, respectively. They are soluble in organic solvents, such as CHCl 3 and Toluene. The absorption thresholds of these two polymers are 500 and 650 nm for PDOCPTPy and PDOCPTPh, respectively. The electronic absorption data suggested that the band gap energy of copolymer will fall in-between the individual homopolymers. This may give a cue on how to prepare soluble conjugated polymers with desirable band gap energy.

Theoretical studies of ground and excited electronic states of complexes M(CO) 4(phen) (M = Cr, Mo, W; phen = 1,10-phenanthroline)

Electronic absorption and emission data of M(CO) 4(phen) complexes, where M = Cr, Mo or W; phen=1,10-phenanthroline are reported using density functional theory (DFT). Electronic absorption spectra of [M(CO) 4(phen)] show a strong band in the visible region of the spectrum, whose metal to ligand charge transfer (MLCT) M → phen and M → CO ax character is indicated by high molar absorptivity. This assignment is investigated by theoretical calculations, which differ from the early interplay of M → L metal to ligand charge transfer MLCT and ligand-filed (LF) electronic transitions, occurring between d-orbitals. The phosphorescence of each complex was identified as a lowest, triplet metal to ligand charge transition (MLCT) around 740 nm.

Synthesis, spectroscopic and electrochemical investigations of supramolecular nickel(II)tetraazaporphyrin complexes

The ( bpy )2RuIIand ( phen )2RuIImoieties were linked to [ Ni ( OBTTAP )]1, periphery through coordinate bonds in order to synthesize cationic di- and pentanuclear complexes 2-5 that were obtained as PF6−salts. They were characterized by IR,1H NMR, UV-vis, and mass spectral data. The electronic absorption, emission and redox data of these bichromophoric systems indicate the presence of a high degree of intercomponent electronic interaction. The position and relative intensities of the Soret and Q bands in these complexes is altered due to peripheral binding of the metal units. The compounds were non-emissive for the Q band excitation but, Soret excitation led to a strong S2emission, observed between 400-450 nm. In cyclic voltammetry, the compounds exhibited one Ru centered oxidation together with one or two OBTTAP centered oxidations. The RuII/ RuIIIoxidations were observed at significantly lower potentials as compared to the corresponding simple maleonitrile-benzylthioether complexes and has been interpretted in terms of weaker d π( S )– d π( Ru ) interactions.

Spectral Luminescence Properties of Potassium Aluminophosphate Glass Doped with Four-Valence Vanadium Complexes

Potassium aluminophosphate glass with the composition 30 K2O-20 Al2O3-50 P2O5 (wt %) doped with four-valence vanadium was prepared and analyzed. Some physicochemical properties of the glass obtained and its absorption and luminescence spectra were investigated at 300 K. The existence of oxovanadium ions in the prepared glass was proved on the basis of the electronic absorption data. The bands observed in the electronic absorption spectrum were assigned to the e→b 1 and e→a 1 transitions. Intense luminescence of four-valence vanadium in the wavelength range 800–1100 nm was found. The average luminescence decay time amounts to 12 μs.

On the action of ozone on methaemoglobin

The reaction of ozone with methaemoglobin in formic acid, 6 M urea solution, NaOH 0.1 M and in acetic/acetate buffer at pH = 5 has been studied. Electronic absorption data, polarimetric and viscometric measurements clearly show that ozone destroys the haeme prosthetic groups of methaemoglobin and causes a strong denaturation of this protein. Furthermore, also by electronic and by FT-IR spectroscopy it is presented the evidence of the oxidation and denaturation of methaemoglobin caused by O 3 of methaemoglobin. The results of the study, and the associated risks and concerns are briefly discussed in the context of the auto-haemotherapy, a medical practice which involves the treatment with ozone and the re-injection of an aliquot of blood taken from a “patient”.

Electronic Spectra and Configuration Interaction of Tm3+ in TmCl63-

Low-temperature electronic absorption and emission data are reported for Tm 3 + at the octahedral site in crystals of Cs 2 NaTmCl 6 . Thirty-seven crystal field levels (total degeneracy 88) of the f 1 2 configuration (totaldegeneracy 91) have been assigned, and in several cases the levels are split due to electron-phonon coupling interactions. The fitting of the energy levels, using the conventional f 1 2 analysis with 12 variable parameters, gives a mean deviation of 53.3 cm - 1 . This is reduced to 9.3 cm - 1 by including the 4f 1 2 np 6 /4f 1 3 np 5 configuration interaction, using 16 variable parameters. The results indicate a tendency for the early members in the series of Ln 3 + ions to interact with the p-electron, and the later members with the p-hole, configurations, following the redox properties of the ions. The interacting configuration is charge-transfer (n = 3) rather than metal ion (n = 5), and the mixing of ligand wave functions with those of the metal ion may be responsible for the unusually strong electron-phonon coupling identified for several electronic states of Tm 3 + in TmCl 6 3-.

Solid‐Phase Synthesis of Oligo(triacetylene)s and Oligo(phenylenetriacetylene)s Employing Sonogashira and CadiotChodkiewicz‐Type Cross‐Coupling Reactions

AbstractWe describe the first polymer‐supported synthesis of poly(triacetylene)‐derived monodisperse oligomers, utilizing Pd0‐catalyzed Sonogashira and CadiotChodkiewicz‐type cross‐couplings as the key steps in the construction of the acetylenic scaffolds. For our investigations, Merrifield resin functionalized with a 1‐(4‐iodoaryl)triazene linker was chosen as the polymeric support (R2; Figure and Scheme 3). The linker selection was made based on the results of several model studies in the liquid phase (Schemes 1 and 2). For the solid‐support synthesis of the oligo(phenylene triacetylene)s 7b–7d, a set of only three reactions was required: i) Pd0‐catalyzed Sonogashira cross‐coupling, ii) Me3Sialkyne deprotection by protodesilylation, and iii) cleavage of the linker with liberation of the generated oligomers (Scheme 5). The longest‐wavelength absorption maxima of the oligo(phenylene triacetylene)s 7a–7d shift bathochromically with increasing oligomeric length, from λmax 337 nm (monomer 7a) to 384 nm (tetramer 7d; Table 2). Based on the electronic absorption data, the effective conjugation length (ECL) of the oligo(phenylene triacetylene)s is estimated to involve at least four monomer units and 40 C‐atoms. π‐Electron conjugation in these oligomers is less efficient than in the known oligo(triacetylene)s 14a–14d (Table 2) due to poor transmittance of π‐electron delocalization by the phenyl rings inserted into the oligomeric backbone. Similar conclusions were drawn from the electrochemical properties of the two oligomeric series as determined by cyclic (CV) and rotating‐disk voltammetry (RDV; Table 3). In sharp contrast to 14b–14d, the oligo(phenylene triacetylene)s 7b–7d are strongly fluorescent, with the highest quantum yield ΦF=0.69 measured for trimer 7c (Table 2). Whereas the Sonogashira cross‐coupling on solid support proceeded smoothly, optimal conditions for alkynealkyne cross‐coupling reactions employing Pd0‐catalyzed CadiotChodkiewicz conditions still remain to be developed, despite extensive experimentation (Scheme 7 and Table 1).

High-frequency and -field EPR spectroscopy of tris(2,4-pentanedionato)manganese(III): investigation of solid-state versus solution Jahn-Teller effects.

High-frequency and -field electron paramagnetic resonance (HFEPR) spectroscopy of a classical coordination complex, Mn(acac)(3) (Hacac = 2,4-pentanedione), has been performed on both solid powder and frozen solution (in CH(2)Cl(2)/toluene, 3:2 v/v) samples. Parallel mode detection X-band EPR spectra exhibiting resolved (55)Mn hyperfine coupling were additionally obtained for frozen solutions. Magnetic susceptibility and field-dependent magnetization measurements were also made on powder samples. Analysis of the entire EPR data set for the frozen solution allowed extraction of the relevant spin Hamiltonian parameters: D = -4.52(2); |E| = 0.25(2) cm(-1); g(iso) = 1.99(1). The somewhat lower quality solid-state HFEPR data and the magnetic measurements confirmed these parameters. These parameters are compared to those for other complexes of Mn(III) and to previous studies on Mn(acac)(3) using X-ray crystallography, solution electronic absorption spectroscopy, and powder magnetic susceptibility. Crystal structures have been reported for Mn(acac)(3) and show tetragonal distortion, as expected for this Jahn-Teller ion (Mn(3+), 3d(4)). However, in one case, the molecule exhibits axial compression and, in another, axial elongation. The current HFEPR studies clearly show the negative sign of D, which corresponds to an axial (tetragonal) elongation in frozen solution. The correspondence among solution and solid-state HFEPR data, solid-state magnetic measurements, and an HFEPR study by others on a related complex indicates that the form of Mn(acac)(3) studied here exhibits axial elongation in all cases. Such tetragonal elongation has been found for Mn(3+) and Cr(2+) complexes with homoleptic pseudooctahedral geometry as well as for Mn(3+) in square pyramidal geometry. This taken together with the results obtained here for Mn(acac)(3) in frozen solution indicates that axial elongation could be considered the "natural" form of Jahn-Teller distortion for octahedral high-spin 3d(4) ions. The previous electronic absorption data together with current HFEPR and magnetic data allow estimation of ligand-field parameters for Mn(acac)(3).

Initial characterization of the ferric H175G cytochrome c peroxidase cavity mutant using magnetic circular dichroism spectroscopy: phosphate from the buffer as an axial ligand

Electronic absorption and magnetic circular dichroism (MCD) data are reported for the H175G cavity mutant of yeast cytochrome c peroxidase (H175G CCP) in its ligand-free ferric state at 4 °C under slightly acidic conditions (pH 5.9). Initial analysis of the MCD spectrum of this state suggested a five-coordinate structure with ligation by an aspartate or glutamate. However, examination of the crystal structure of wild-type CCP revealed no plausible carboxylate-containing amino-acid residues close enough to the heme iron to serve as the ligand. An alternative interpretation of the MCD data is that the phosphate ion from the buffer is bound to the ferric heme iron. A phosphate group is similar in electronic character to a carboxylate and could give an MCD signal similar to that of a carboxylate-bound heme. Phosphate coordination to the ferric heme iron, albeit trans to imidazole, has recently been seen in a 2.0-Å-resolution crystal structure of imidazole-bound ferric H175G CCP in phosphate buffer [Biochemistry 40 (5) (2001) 1265]. To investigate the validity of phosphate ligation, the pH 5.9 species in the 100 mM phosphate buffer was exchanged into the pH 5.9, 100 mM MES (2-[ N-morpholino]ethanesulfonic acid) buffer. The MCD spectrum of the species in the MES buffer was spectrally distinct from that in the phosphate buffer, indicating that the original spectrum depends on the presence of a phosphate ion. We conclude that in the phosphate buffer, the exogenous ligand-free ferric state of the H175G CCP cavity mutant is actually coordinated by a phosphate ion from the buffer.

Near Infrared Optical Absorption of Gold Nanoparticle Aggregates

The reduction of HAuCl4 by Na2S has been reported to produce gold nanoparticles with an optical absorption in the near-infrared along with its characteristic absorption in the visible. The optical resonances in the visible are due to the gold surface plasma, which are a function of the geometry of the particles. The near-infrared absorption had been attributed to the formation of Au2S/Au core/shell structures. In this report we present new electronic absorption, electron microscopy, and X-ray absorption data in several systems to show that the near-infrared absorption does not involve core/shell structures. We further suggest that the near-infrared adsorption is most likely the result of the formation of aggregates of gold nanoparticles. The identification of the origin of the near-infrared resonance is critical in understanding the optical properties of metal nanoparticle systems.

Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus.

The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli. The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth. The as-purified PfMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis. This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes. When an additional 2 equiv of Zn(II) was added to the as-purified PfMetAP-II, no activity could be detected. The combination of these data with previously reported whole cell studies on EcMetAP-I further supports the suggestion that the in vivo metal ion for all MetAP's is Fe(II). Both Co(II)- and Fe(II)-loaded PfMetAP-II showed similar substrate specificities to EcMetAP-I. Substrate binding was largely affected by the amino acid in the P1 position and the length of the polypeptide. The substrates MSSHRWDW and MP-p-NA showed the smallest K(m) values while the substrates MGMM and MP-p-NA provided the highest turnover. The catalytic efficiency (k(cat)/K(m)) of PfMetAP-II for MP-p-NA at 30 degrees C was 799 500 and 340 930 M(-1) s(-1) for Co(II)- and Fe(II)-loaded PfMetAP-II, respectively. Maximum catalytic activity was obtained with 1 equiv of Co(II) or Fe(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 50 +/- 15 and 20 +/- 15 nM for Co(II)- and Fe(II)-substituted PfMetAP-II, respectively. Electronic absorption spectral titration of a 1 mM sample of apo-PfMetAP-II with Co(II) provided a dissociation constant of 0.35 +/- 0.02 mM for the second metal binding site, a 17500-fold increase compared to the first metal binding site. The electronic absorption data also indicated that both Co(II) ions reside in a pentacoordinate geometry. PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be approximately 85 degrees C at pH 7.5 in 25 mM Hepes and 150 mM KCl buffer. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the k(cat) values and was found to be linear over the temperature range 25-85 degrees C, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature. Co(II)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively). The thermodynamic parameters calculated at 25 degrees C are as follows: DeltaG++ = 46.23 kJ/mol, DeltaH++ = 10.79 kJ/mol, and DeltaS++ = -119.72 J.mol(-1).K(-1) for Co(II)-loaded PfMetAP; DeltaG++ = 46.44 kJ/mol, DeltaH++ = 16.94 kJ/mol, and DeltaS++ = -99.67 J.mol(-1).K(-1) for Fe(II)-loaded PfMetAP. Interestingly, at higher temperatures (> 50 degrees C), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 degrees C) than Co(II)-loaded PfMetAP-II.

Definitive evidence for monoanionic binding of 2,3-dihydroxybiphenyl to 2,3-dihydroxybiphenyl 1,2-dioxygenase from UV resonance Raman spectroscopy, UV/Vis absorption spectroscopy, and crystallography.

Ultraviolet resonance Raman spectroscopy (UVRRS), electronic absorption spectroscopy, and X-ray crystallography were used to probe the nature of the binding of 2,3-dihydroxybiphenyl (DHB) to the extradiol ring-cleavage enzyme, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD; EC 1.13.11.39). The lowest lying transitions in the electronic absorption spectrum of DHBD-bound DHB occurred at 299 nm, compared to 305 nm for the monoanionic DHB species in buffer. In contrast, the corresponding transitions in neutral and dianionic DHB occurred at 283 and 348 nm, respectively, indicating that DHBD-bound DHB is monoanionic. These binding-induced spectral changes, and the use of custom-designed optical fiber probes, facilitated UVRR experiments. The strongest feature of the UVRR spectrum of DHB was a Y8a-like mode around 1600 cm(-1), whose position depended strongly on the protonation state of the DHB. In the spectrum of the DHBD-bound species, this feature occurred at 1603 cm(-1), as observed in the spectrum of monoanionic DHB. Raman band shifts were observed in deuterated solvent, ruling out dianionic binding of the substrate. Thus, the electronic absorption and UVRRS data demonstrate that DHBD binds its catecholic substrate as a monoanion, definitively establishing this feature of the proposed mechanism of extradiol dioxygenases. This conclusion is supported by a crystal structure of the DHBD:DHB complex at 2.0 A resolution, which suggests that the substrate's 2-hydroxyl substituent, and not the 3-hydroxyl group, deprotonates upon binding. The structural data also show that the aromatic rings of the enzyme-bound DHB are essentially orthogonal to each other. Thus, the 6 nm blue shift of the transition for bound DHB relative to the monoanion in solution could indicate a conformational change upon binding. Catalytic roles of active site residues are proposed based on the structural data and previously proposed mechanistic schemes.

Interactions of bismuth with human lactoferrin and recognition of the Bi(III)-lactoferrin complex by intestinal cells.

Several bismuth compounds are currently used as antiulcer drugs, but the mechanism of action still remains unclear. The antimicrobial activity of Bi(III) complexes toward Gram-negative bacteria is reported to be dependent on the iron uptake system [Domenico, P., et al. (1996) J. Antimicrob. Chemother. 38, 1031-1040]. Electronic absorption and 13C NMR spectroscopic data show that Bi(III) binds to human lactoferrin at the specific Fe(III) sites along with either carbonate or oxalate as the synergistic anion. The uptake of Bi(III) by apo-hLF was rapid [minutes in 10 mM Hepes buffer and 5 mM bicarbonate (pH 7.4)], and almost equal in both lobes. The presence of ATP facilitates the release of Bi(III) from the Bi2-hLF complex when the pH is lowered. The Bi2-hLF complex blocked the uptake of the radiolabeled 59Fe-hLF complex into rat IEC-6 cells. Surprisingly, apo-hLF (but not apotransferrin) was almost as effective in blocking 59Fe uptake as bismuth-loaded lactoferrin. These results suggest that Bi(III)-loaded hLF might be recognized by the lactoferrin receptor and be taken up into cells.

Symmetry states of metalloporphyrin π-cation radicals, models for peroxidase compound I

Oxoferryl porphyrin π-cation radical active sites of compound I intermediates which are found in enzymes such as peroxidases and catalases have been extensively modeled by oxidized synthetic metalloporphyrins. The electronic symmetry states of these compounds were initially assigned on the basis of electronic absorption data. In recent years new experimental and theoretical results have become available which have led to a re-evaluation and modification of the original assignments. A historical perspective of these developments is provided in the context of recent NMR, resonance Raman, and other spectroscopic data and theoretical calculations for the synthetic models and enzymatic systems.

Spectroscopic properties of porphyrin dimers incorporating phenylenevinylene linkers

A series of free-base, zinc and mixed free-base-zinc porphyrin dimers were investigated using electrochemistry, electronic absorption, resonance Raman and emission studies. The spectroelectrochemistry of these compounds was also examined. The electronic absorption and resonance Raman data suggest that the two porphyrins in the dimer are behaving as independent chromophores with limited communication. However, emission studies show that energy transfer occurs between the two units. The linking unit is seen to have limited influence on the properties of chromophores.

Spectroscopic and electrochromical properties of metallophthalocyanines in silicate bulks and thin films prepared by the sol–gel method

Magnesium phthalocyanine (MgPc) and praseodymium bisphthalocyanine (Pr(Pc) 2) have been entrapped in bulks and thin films of porous silicate xerogels prepared via the sol–gel method. The electronic absorption data obtained for the complexes immobilized in bulk sol–gel matrices indicate the formation of complex equilibria between their monomeric forms and respective agglomerates. Thin xerogel films doped with metallophthalocyanines and connecting two slides of transparent ITO electrodes display changes in the absorption spectra caused by varying of the applied voltage. These changes appear to be reversible in certain ranges of the applied voltages.

Metal-to-ligand charge-transfer excited-states in binuclear copper(I) complexes. Tuning MLCT excited-states through structural modification of bridging ligands. A resonance Raman study

The resonance Raman spectra of the ground- and lowest excited-states for a series of binuclear copper(I) complexes with bridging ligands based on 2,3-di-(2-pyridyl)quinoxaline have been measured. Analyses of the ground-state resonance Raman spectra show strong enhancement of an inter-ring stretching mode and quinoxaline-based modes; consistent with the Frank–Condon state having bonding changes about the copper(I) centre and on the quinoxaline ring system. The electronic absorption data also suggest the initially formed MLCT-state is localised towards the quinoxaline ring system. The excited-state resonance Raman spectroscopy shows similar spectral features for complexes which have substituent changes at the pyridyl rings of the bridging ligand. They show spectra associated with the radical anion of the bridging ligand. This is consistent with the formation of an excited-state which is metal-to-ligand charge-transfer in nature and where the radical anion is localised on the quinoxaline ring system of the bridging ligand.

Inhibition of the aminopeptidase from Aeromonas proteolytica by l-leucinethiol: kinetic and spectroscopic characterization of a slow, tight-binding inhibitor–enzyme complex

The peptide inhibitor l-leucinethiol (LeuSH) was found to be a potent, slow-binding inhibitor of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potency ( K I *) of LeuSH was 7 nM while the corresponding alcohol l-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency ( K I=17 μM). These data suggest that the free thiol is likely involved in the formation of the E·I and E·I * complexes, presumably providing a metal ligand. In order to probe the nature of the interaction of LeuSH and LeuOH with the dinuclear active site of AAP, we have recorded both the electronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] in the presence of both inhibitors. In the presence of LeuSH, all three Co(II)-substituted AAP enzymes exhibited an absorption band centered at 295 nm, characteristic of a S→Co(II) ligand-metal charge-transfer band. In addition, absorption spectra recorded in the 450 to 700 nm region all showed changes characteristic of LeuSH and LeuOH interacting with both metal ions. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that, in a given enzyme molecule, LeuSH interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified μ-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon binding LeuSH. EPR spectra of [CoCo(AAP)]-LeuSH, [ZnCo(AAP)]-LeuSH, and [Co_(AAP)]-LeuSH were also recorded at lower temperature (3.5–4.0 K) and high microwave power (50–553 mW). These signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at g eff∼6.5 that is characteristic of thiolate-Co(II) interactions. Combination of the electronic absorption and EPR data indicates that LeuSH perturbs the electronic structure of both metal ions in the dinuclear active site of AAP. Since the spin–spin interaction seen in resting [CoCo(AAP)] is abolished upon the addition of LeuSH, it is unlikely that a μ-S(R) bridge is established.

Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4°C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP(MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met–Lys–Thr (MKT) at the N-terminus, CCP(MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP(MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP(MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP(MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (≤5%) in the spectrum of CCP(MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP ( λ CT=637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP(MKT) ( λ CT=637 nm) solved at 2.0 Å resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.