The effect of iron displacement out of the porphyrin plane on the resonance Raman spectra of heme proteins and iron porphyrins.

The causes of the strong coupling of the iron-histidine vibration to the Soret resonance in the resonance Raman spectra of deoxyhemoglobin, myoglobin, and peroxidase are explored, using the vibronic theory. It is shown that the extent of the iron displacement out of the plane of the porphyrin nitrogens is the main structural parameter controlling the Fe-NHis band features, such as the dependence of its frequency and intensity on the protein conformation and number of the axial ligands, time evolution after the photolysis of the diatomic complexes of the proteins under consideration, and inverse relationship between the changes Fe-NHis and v4 porphyrin breathing mode frequencies.     

Visible absorption spectra of quantum mixed-spin ferric heme proteins.

Recently, it has been shown that the magnetic data for Chromatium ferricytochrome c' at pH 7 are consistent with quantum mechanically (as distinguished from thermally) mixed mid-spin (S = 3/2) and high-spin (S = 5/2) heme. Visible absorption spectra of the protein measured at 77 degrees K and 293 degrees K, pH 7, show peaks at 400, 490, and 632 nm. The observation of a 630 nm band in quantum mixed-spin heme spectra, and the spin state-dependence of the band intensity, are discussed in the context of the iron-ligand structure for quantum mixed-spin heme inferred from magnetic data.     

Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins

The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.     

Novel rhodium porphyrin derivatives

Hydrated rhodium trichloride reacts with porphyrins in dimethylformamide to give bis-dimethylamino (DMA) derivatives of general formula [RhP(DMA)₂- X]. Modifications of the spectral properties caused by anion exchange are discussed.     

The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra.

Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.     

The impact of altered protein-heme interactions on the resonance Raman spectra of heme proteins. Studies of heme rotational disorder

Heme proteins that have been reconstituted with certain hemins may contain substantial fractions of a minor component in which the orientation of the heme in the folded pocket differs from the major ("native") conformation by a 180 degrees rotation about the alpha-gamma meso axis. In fact, this minor component has also been shown to exist in some native proteins, including several mammalian globins. While resonance Raman spectroscopy has emerged as a powerful probe of active site structure of heme proteins, no systematic study has yet been undertaken to elucidate the specific spectral changes associated with this disorder. In the present work, combined analyses of the temporal behavior of both NMR and RR data sets have been completed to permit the extraction of a unique RR spectrum for the disoriented form, documenting rather dramatic changes associated with this rotational disorder. In addition, the use of protohemes bearing selectively deuterated peripheral methyl groups has permitted the association of the observed modes with specific fragments of the heme residing in the reversed orientation. The studies conducted here clearly illustrate the exquisite sensitivity of low frequency heme deformation modes to altered protein-heme interactions.     

Solvent effects on fluorescence properties of protochlorophyll and its derivatives with various porphyrin side chains

Fluorescence spectra and fluorescence lifetimes of protochlorophyll (Pchl) were measured in organic solvents having different physical and chemical properties and were analyzed taking into account the nonspecific (dependent on bulk solvent parameters), and specific (e.g. H bonds, Mg coordination) solvent–solute interactions. The energy of the fluorescence emission band decreased, while the Stokes shift increased for increasing solvent orientation polarizability, which is a function of both the dielectric constant (ε) and the refractive index (n). The extent of the dependence of the Stokes shift on solvent orientation polarizability was higher in protic (i.e. those able to form hydrogen-binding) than in aprotic solvents. High value of the Stokes shift was also observed in pyridine and methanol, i.e. in solvents hexacoordinating the central Mg atom. The fluorescence decay of Pchl was monoexponential in all of the investigated solvents. The fluorescence lifetime decreased for increasing solvent orientation polarizability from 5.5 ± 0.1 ns in 1,4-dioxane to 3.3 ± 0.1 ns in methanol. Longer lifetime values were observed in the case of aprotic solvents than in protic solvents. The hexacoordination of Mg had no effect on the fluorescence lifetime. The present data are discussed with respect to results found for protochlorophyllide (Pchlide) (My?liwa-Kurdziel et al. in Photochem Photobiol 79:62–67, 2004), and they indicate that the presence of phytol chain in the porphyrin ring influences the spectral properties of the whole chromophore. This is the first complex analysis comparing the fluorescence emission and fluorescence lifetimes of purified Pchl and Pchlide.     

Lanthanide porphyrin probes of heme proteins. Insertion of ytterbium (III) mesoporphyrin IX into apomyoglobin.

Apomyoglobin has been reconstituted with the lanthanide porphyrin complex, ytterbium(III)mesoporphyrin IX. The reconstituted material exhibits absorption and magnetic circular dichroic spectra significantly different from those of the ytterbium porphyrin itself. The sizeable, positive extrinsic Cotton effect in the Soret band of Yb-mesoporphyrin IX induced by the interactions with the globin indicates that the lanthanide porphyrin complex occupies the heme crevice.     

Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures

Tryptophan fluorescence lifetimes were analyzed for three proteins: human serum albumin, bovine serum albumin, and bacterial luciferase, which contain one, two, and seven tryptophan residues, respectively. For all of the proteins, the fluorescence decays were fitted by three lifetimes: τ₁ = 6–7 ns, τ₂ = 2.0–2.3 ns, and τ₃ ≤ 0.1 ns (the native state), and τ₁ = 4.4–4.6 ns, τ₂ = 1.7–1.8 ns, and τ₃ ≤ 0.1 ns (the denatured state). Corresponding decay-associated spectra had similar peak wavelengths and spectrum half-widths both in the native state (\(\lambda _{\max }^{{\tau _1}} = 324nm\), \(\lambda _{\max }^{{\tau _2}} = 328nm\), and \(\lambda _{\max }^{{\tau _3}} = 315nm\)), and in the denatured state (\(\lambda _{\max }^{{\tau _1}} = 350nm\), \(\lambda _{\max }^{{\tau _2}} = 343nm\), and \(\lambda _{\max }^{{\tau _3}} = 317nm\)). The differences in the steady-state spectra of the studied proteins were accounted for the individual ratio of the lifetime component contributions. The lifetime components were compared with a classification of tryptophan residues in the structure of these proteins within the discrete states model.     

Carbon monoxide-driven reduction of ferric heme and heme proteins

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.     

The metal complexes of N-confused porphyrin as heme model compounds

Recently, metal complexes of the isomers and analogs of porphyrin have become important model compounds for heme enzymes and proteins. While the chemistry of metalloporphyrins as heme models still attracts attention, the isomers and analogs of porphyrins provide insight into the biological choice of porphine as the macrocycle of choice and also help model reactive intermediates, such as high valent oxidation states. In this mini-review, we discuss the heme-relevant chemistry of N-confused porphyrin, an isomer of porphyrin with an inverted pyrrole ring, and focus on the chemistry of manganese, iron, and cobalt. The metallation chemistry of this macrocycle is more diverse than normal porphyrin, and involves tautomerization, C-H bond activation, the Lewis basicity of the external nitrogen, and issues with nucleophilic sensitivity. Despite the challenges posed by N-confused porphyrin, significant progress has been made toward generating heme-model complexes with this macrocycle.     

Endonuclease-like activity of heme proteins

Heme proteins, metmyoglobin, methemoglobin, and metcytochrome c showed unusual affinity for double-stranded DNA. Calorimetric studies show that binding of methemoglobin to calf thymus DNA (CTDNA) is weakly endothermic, and the binding constant is 4.9+/-0.7x10(5) M(-1). The Soret absorption bands of the heme proteins remained unchanged, in the presence of excess CTDNA, but a new circular dichroic band appeared at 210 nm. Helix melting studies indicated that the protein-DNA mixture denatures at a lower temperature than the individual components. Thermograms obtained by differential scanning calorimetry of the mixture indicated two distinct transitions, which are comparable to the thermograms obtained for individual components, but there was a reduction in the excess heat capacity. Activation of heme proteins by hydrogen peroxide resulted in the formation of high valent Fe(IV) oxo intermediates, and CTDNA reacted rapidly under these conditions. The rate was first-order in DNA concentration, and this reactivity resulted in DNA strand cleavage. Upon activation with hydrogen peroxide, for example, the heme proteins converted the supercoiled pUC18 DNA into nicked circular and linear DNA. No reaction occurred in the absence of the heme protein, or hydrogen peroxide. These data clearly indicate a novel property of several heme proteins, and this is first report of the endonuclease-like activity of the heme proteins.     

Determination of tyrosine and tryptophan in proteins by the absorption spectra of derivatives

The contents of tyrosine and tryptophan were determined by the method based on the first derivatives of the proteins absorption spectra within the alkali pH. The advantages of the method are as follows: a much better resolution of tyrosine and tryptophan spectral maxima located at 293 and 307 nm, respectively, which allows the precision of their assay to be increased, especially in case of a small amount of one amino acid and relative abundance of the other and high turbidity of the preparations; the account of the cystine absorption is not necessary; possibility to study proteins containing the chromophore prosthetic groups.     

Analysis of log-normal components of fluorescence spectra of prodan and acrylodan bound to proteins

Steady-state fluorescence spectra of prodan and acrylodan covalently bound to cystein residue of Lys-Cys-Phe tripeptide in solvents of different polarity were analyzed. It was shown that the shape of spectral bands is well described by a log-normal function. Linear relations between three shape-determining parameters of the log-normal function (namely, the positions of spectral maximum and two half-maximum amplitudes) were revealed and evaluated for both fluorophores. This finding enabled us to present the shape of spectral bands of these fluorophores in any environment as analytical log-normal functions depending on only two parameters, the maximum position and the peak amplitude. The empirical uniparametric log-normal curve was used for the analysis of composite fluorescence spectra of prodan bound to bovine serum albumin and acrylodan covalently attached to actin or subfragment 1 of myosin.