Biomedical subjects

J M Vanderkooi

Publications and source records attributed to J M Vanderkooi.

At least 19 recordsLinked to original sources

PubMed1999

Resonance Raman investigation of nickel microperoxidase-11.

Resonance Raman and UV-visible absorption spectra show that nickel(II) microperoxidase-11 (NiMP-11) is four-coordinate in aqueous solution in the pH range from 1.0 to 13.0. In aqueous solutions of NiMP-11 in the absence of cetyltrimethylammonium bromide (CTAB), NiMP-11 is aggregated. In CTAB micellar solutions, where aggregation of NiMP-11 does not occur, the Raman spectra of NiMP-11 are similar to that of nickel(II) cytochrome c (NiCyt-c). The presence of the peptide segment shifts the equilibrium heavily in favor of the nonplanar form, just as does the entire protein component in the case of NiCyt-c. This further elucidates the structural mechanism by which the protein segment ruffles the heme, most likely modulating the redox potential as indicated for the cytochromes c3 [Ma, J.-G., et al. (1998) Biochemistry 37, 12431-12442]. Furthermore, the hydrophobic environment that is provided by the CTAB micelle is found to be crucial to the native folding of the pentapeptide and formation of two hydrogen bonds in the peptide backbone. These two H-bonds act to contract the peptide segment exerting the force on the macrocycle that causes the ruffling and makes the redox potential more negative than if the heme were to remain planar. The structure of the heme and pentapeptide may also be associated with redox-linked triggering of the formation and release of cytochrome-protein complexes.

Cytochrome c Group↗

PubMed1999

The connection domain is implicated in metalloporphyrin binding and inhibition of HIV reverse transcriptase.

We have shown that heme and zinc protoporphyrin inhibit both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs) and, in combination with other nucleoside and non-nucleoside inhibitors, exert an additive effect on HIV-1 RT inhibition. Screening of a phage peptide library against heme resulted in the isolation of a peptide with sequence similarity to sequence 398-407 from the connection subdomain of both HIV-1 and HIV-2 RTs, suggesting that this highly conserved region of HIV RTs corresponds to the binding site for metalloporphyrins and a new site for inhibition of enzyme activity. Inclusion of a synthetic peptide corresponding to the exact sequence 398-407 of HIV-1 RT in RT inhibition assays had a protective effect on metalloporphyrin inhibition, as it was able to reverse the inhibitory effect of both metalloporphyrins on HIV-1 RT activity. Furthermore, intrinsic fluorescence assays indicated that these metalloporphyrins bind to synthetic peptide 398-407 as well as to intact dimeric HIV-1 RT. The identification of this novel inhibition site will help to expand our understanding of the mode of action of metalloporphyrins in RT inhibition and will assist in the design and development of more potent metalloporphyrin RT inhibitors for the management of HIV infection.

Amino Acid Sequence↗

PubMed1999

Protonation of porphyrin in iron-free cytochrome c: spectral properties of monocation free base porphyrin, a charge analogue of ferric heme.

Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers, a charge typically exists at the prosthetic group within the interior. How a protein accommodates a buried charge and the effect of redox changes on protein stability are thermodynamically related problems. To examine these problems in cytochrome c, the metal-free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their characteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately alpha-helical structure, although the carboxyl groups of the dicarboxylic amino acids become protonated at lower pH. The monocation porphyrin form (which has not been previously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two protonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded protein, and 4.4 and 3.1 for the unfolded protein. These values indicate that the protection of the polypeptide chain for protonation is approximately 3 kcal.

Amino Acid Sequence↗

PubMed1998

The protein state of matter.

Three ways are generally used to visualize proteins: (1) a static model in which the atomic positions are defined, (2) a dynamic model taking into account fluctuations, and (3) a reactive model that reflects the internal and external electric fields of the molecule. The properties of chromophoric prosthetic groups can be probed by optical spectroscopy, and when high resolution techniques are used, the results reveal information about the local electric fields in proteins, as influenced and determined by atomic positions and dynamics.

Electricity↗

PubMed1998

Fluorescence line narrowing applied to the study of proteins.

Fluorescence line narrowing is a high resolution spectroscopic technique that uses low temperature and laser excitation to optically select specific subpopulations from the inhomogeneously broadened absorption band of the sample. When applied to the study of fluorescent groups in proteins one can obtain vibronically resolved spectra, which can be analyzed to give information on spectral line shapes, vibrational energies of both the ground and excited state molecule, and the inhomogeneous distribution function of the electronic transitions. These parameters reveal information about the chromophoric prosthetic group and the protein matrix and are functions of geometric strains and local electric fields imposed by the protein. Examples of the use of fluorescence line narrowing are discussed in investigations of heme proteins, photosynthetic systems and tryptophan-containing proteins.

Hemeproteins↗

PubMed1998

Zinc-substituted hemoglobins: alpha- and beta-chain differences monitored by high-resolution emission spectroscopy.

The absorption and emission properties of hybrid Zn-substituted human hemoglobin (Hb) were used to monitor differences in interaction between the porphyrin and the polypeptide chain for the two subunits. Although alpha-substituted (alpha-ZnHb), beta-substituted (beta-ZnHb), or totally substituted Hb all show optical properties characteristic of Zn porphyrins, the spectra are also indicative of specific interactions between the polypeptide chain and the porphyrin. The Q0,0 absorption band of alpha-ZnHb at 5 K shows a splitting of approximately 300 cm-1, comparable to the largest split ever reported for a heme protein. This value is approximately 140 cm-1 for beta-ZnHb. The possible origin of the split is discussed in terms of the local electric field imposed by the amino acids of the respective heme pockets, different configurations of the porphyrin, and/or influences of the liganding histidine. The Zn derivatives show quasiline spectra under fluorescence line narrowing conditions, and the resolved excitation spectrum reveals differences in the vibrational levels of the Zn porphyrin in the two subunits. Broad underlying emission in the fluorescence line-narrowed emission spectrum can be accounted for, in part, by the existence of the two closely spaced electronic origins and also by the extent of phonon coupling between the porphyrin and the protein matrix.

Crystallography, X-Ray↗

PubMed1998

Protein-induced changes in nonplanarity of the porphyrin in nickel cytochrome c probed by resonance Raman spectroscopy.

The influence of the protein on the nonplanarity of the macrocycle for nickel(II)-reconstituted cytochrome c (NiCyt-c) has been investigated with pH-dependent resonance Raman and UV-visible absorption spectroscopy and molecular mechanics calculations. The spectra reveal that NiCyt-c near neutral pH has axially coordinated Ni, but below pH 3 and above pH 12, four-coordinate species predominate. The shape of the structure-sensitive Raman line nu10 of NiCyt-c is asymmetric and broad and it changes with pH. This broad line can be decomposed well into at least two sublines, a low-frequency line that results from a nonplanar conformer and a high-frequency line that arises from a nearly planar conformer. Upon lowering the pH from 3.0 to 1.0, the amount of the nonplanar conformer decreases relative to that of the planar conformer. The decreased nonplanarity can be accounted for in terms of the disruption of a hydrogen-bonding network in the peptide backbone upon lowering the pH. Molecular mechanics (MM) calculations on iron(III) and nickel(II) microperoxidase 5 (MP-5) as well as some model heme derivatives have been carried out in order to locate the part of the protein that causes the heme distortion observed in the X-ray crystal structures of cytochromes c. The energy-optimized structures of MP-5 and the model compounds were analyzed using the normal-coordinate structural decomposition method to specify and quantify the out-of-plane macrocyclic distortions. MM calculations for MP-5 show that two hydrogen bonds formed between the amide groups in the peptide backbone are important in maintaining the ruffled deformation of the macrocycle. All evidence presented supports the hypothesis that the nonplanar distortion of the porphyrin of cytochromes c is largely maintained by a relatively small protein segment including the cysteines, the amino acids between the cysteines, and the adjacent histidine ligand. Hydrogen bonding within the backbone of this segment is important in maintaining the conformation of the peptide that induces the porphyrin distortion.

Binding Sites↗

PubMed1998

Effect of charge interactions on the carboxylate vibrational stretching frequency in c-type cytochromes investigated by continuum electrostatic calculations and FTIR spectroscopy.

The FTIR spectra of the asymmetric carboxylate absorption region of three c-type cytochromes--namely horse heart, yeast and bonito cytochromes c--as well as continuum electrostatic calculations performed on their respective protein matrices, show that these combined methods can target specific protein regions and yield pertinent protein charge information that correlates with the observed spectral data. Deconvolution of the IR carboxylate stretch frequency region (1525-1675 cm-1) in the three cytochromes yield different v(oco)a distributions. In the case of the bonito cytochrome c carboxylates, two v(oco)a populations are clearly distinguishable in the deconvoluted spectra--which is not the case for the more complex v(oco)a deconvolutions of the other two cytochromes. The frequency distributions of the calculated potentials are consistent with the experimental observations and we conclude that the IR carboxylate absorption in proteins can be modified by the electrostatic environment.

Animals↗

PubMed1998

The photoexcited triplet state as a probe of chromophore-protein interaction in myoglobin.

The photoexcited metastable triplet state of Mg(2+)-mesoporphyrin IX (MgMPIX) or Mg(2+)-protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg2+ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8-110 K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MGPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is >> 150 cm-1, suggesting symmetry breaking from the anisotropy of intermal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527-16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q0.0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg2+ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.

Animals↗

PubMed1998

Microperoxidase-11: molecular dynamics and Q-band excited resonance Raman of the oxidized, reduced and carbonyl forms.

Resonance Raman spectra with Q-band excitation are reported for microperoxidase-11, the cytochrome c analog. Spectra were acquired in the mid-frequency range for the oxidized, and reduced forms of the undecapeptide, as well as for the imidazole and carbonyl complexes. Oxidation and spin state marker bands of the undecapeptides are consistent with a six-coordinate, low spin iron in both oxidation states. Porphyrin core size correlations yield a porphyrin-centre to pyrrole-nitrogen distance of 2.00 A for MP11, suggestive of a six-coordinate species in a distorted heme environment. Molecular dynamics results show that the non-planarity of the heme of the parent cytochrome is conserved in the microperoxidase and its carbonmonoxy analog.

Carbon Monoxide↗

PubMed1997

Surface of cytochrome c: infrared spectroscopy of carboxyl groups.

The carboxylate groups of organic acids give strong absorption in the infrared between approximately 1550 and 1650 cm-1. For acetate and chloroacetate derivatives, the infrared (IR) frequency of the carboxylate antisymmetric stretching mode (v(a)OCO) is related to the square root of the pK of the acid, with a shift of approximately 20 cm-1 to higher frequency for a pK drop in the range 5-3. It follows that v(a)OCO may respond to conditions on the protein surface. In this paper, the IR amide I' and carboxylate absorptions of cytochrome c from horse, yeast, and tuna are compared with model compounds such as Val-Glu and microperoxidase-11, the 11 amino acid fragment of horse cytochrome c containing the covalently bound heme. For microperoxidase-11, the contribution from all four carboxylates can be accounted for and the 1567 cm-1 absorption is assigned to the heme propionates. For the proteins, the carboxylate absorption band is inhomogeneous, i.e., there is a distribution of frequencies. Both the amide I' and carboxylate bands are sensitive to protein conformation as shown by their different pH, salt, and redox dependence.

Amino Acid Sequence↗

PubMed1997

Conformational effects of calcium release from parvalbumin: comparison of computational simulations with spectroscopic investigations.

The effect of Ca2+ binding to parvalbumin was monitored by probes of conformation including absorption, fluorescence, circular dichroism (CD), infrared (IR) spectroscopy and differential scanning calorimetry. These experimental studies were compared with molecular dynamics computations on the structures of the Ca-bound and Ca-free forms of cod parvalbumin. The UV CD spectra show that removal of calcium results in a decrease in the alpha-helical content of the protein. The IR amide I' and III' regions are very much affected by Ca removal and are indicative of significant perturbation of secondary structure. The fluorescence of tryptophan, the IR markers, and UV ellipticity all show changes with temperature, pointing to a lowering of protein stability upon Ca removal. These results are consistent with the structures obtained for both the Ca-bound and Ca-free proteins after 200 ps of solvated molecular dynamics simulations which show a decrease in the secondary structure upon Ca removal.

Calcium↗

PubMed1997

Fluorescence line narrowing spectroscopy: a tool for studying proteins.

Perhaps the most important contribution of FLN is that it provides an experimental approach to relate physical changes in the protein to predicted dynamical behavior. It is clear that the sample is inhomogeneously broadened in a continuous manner, consistent with the damped motion of proteins. At the same time configurational substates can be selected, suggesting that there is indeed a hierarchy of protein motion and structure. As yet, identification of the structure, and relating it to the spectra, has not been achieved. It is clear that the electric field exerted by neighboring atoms shifts the electronic transition, and the inhomogeneity is greater when the surrounding disorder is greater. The inhomogeneity for the chromophore in the protein is dependent on the protein conformation and is intermediate between that of a crystal and a glass. The phonon coupling also depends on the chromophore and the protein. Fluorescence line narrowing provides in addition ground- and excited-state vibrational frequencies, thereby allowing for structural differences between the excited-state and the ground-state molecule to be detected.

Animals↗

PubMed1996

Spectral splitting in the alpha (Q0,0) absorption band of ferrous cytochrome c and other heme proteins.

The alpha or Q0,0 absorption band of horse iron(II) cytochrome c splits and shifts to the blue as temperature decreases over the temperature range of 290-10 K. At room temperature, its maximum is at 18 150 cm-1 and the spectral width is 273 cm-1, whereas at 10 K, the two bands of the Q0,0 transition occur at 18 364 and 18 253 cm-1 and the width of the lowest-energy band is 96 cm-1. Temperature dependent splitting also occurs for zinc cytochrome c, a derivative in which Fe has been replaced by Zn; at 10 K, the peaks in the Q0,0 band region occur at 17 106 and 16 996 cm-1. The peak positions are independent of the cryosolvent (aqueous ethylene glycol or glycerol mixtures). The splitting of the Q0,0 band seen in the protein (approximately 110 cm-1 for iron and zinc cytochrome c) is comparable to the crystal field splitting observed for metalloporphyrins in mixed crystals. In contrast, the Q0,0 band of zinc coproporphyrin III in a glassy solvent (dimethylformamide/ethylene glycol) or in poly(vinyl chloride) shows a blue shift with temperature decrease but no evidence of Q0,0 splitting. Available spectral data show that the Q0,0 band is composed of two nearly degenerate electronic transitions and the split is due to the asymmetry in the heme pocket of the protein that arises from the surrounding polypeptide chain. This asymmetry results in the stabilization of one form of the excited state over the other, according to a Jahn-Teller mechanism.

Animals↗

PubMed1996

Infrared spectroscopy of the cyanide complex of iron (II) myoglobin and comparison with complexes of microperoxidase and hemoglobin.

The cyanide complex of FeIIMb prepared and maintained at temperatures below 0 degrees C is sufficiently stable to permit spectroscopic characterization and allow comparison with free HCN and other ferric and ferrous CN complexes. The visible absorption spectrum of FeIIMb-CN has a split alpha band maxima at 571 and 563 nm, suggesting distortion in the x-y plane of the porphyrin. FeIIMb-CN, like the CO complex, was found to be optically active by circular dichroism. The C-N stretching frequencies for the CN-ferrous complexes are very sensitive to parameters within the heme pocket. The values are as follows: FEIIMb at pH 8, 2057 cm-1 with a shoulder appearing at 2078 cm-1 at pH 5.6; FeIIMp, 2034 cm-1. In contrast, the frequencies for C-N stretch differ little among ferric heme complexes, ranging from 2123 to 2125 cm-1 for myoglobin, hemoglobin, and microperoxidase. These values compare with free HCN (2094 cm-1) or CN- (2080 cm-1). Quantum chemical modeling of the neutral iron-porphyrin complex with imidazole and cyanide and of its anion was used to explain the effects of the cyanide coordination and of iron reduction on the C-N stretching frequencies. The lower nu C-N for FeIIMb-CN relative to the ferric complex is attributed to the appearance of additional electron density on all the anti-bonding CN orbitals. The extra electron density was also used to explain that the band width of C-N stretching mode was greater in the ferrous complexes than in the ferric complex. Finally, the calculation shows that sigma donation weakens the Fe-C bond, in qualitative agreement with the spontaneous dissociation of CN- from FeIIMb at -5 degrees C. The sensitivity of CN complexes of ferrous heme proteins to the heme pocket environment and the ability to correlate spectroscopic parameters with calculated electron density suggest that infrared spectroscopy of the CN ligand is an appropriate tool to study ferrous heme proteins.

Animals↗

PubMed1996

Spectroscopic characterization of bendazac and benzydamine: possible photochemical modes of action.

The involvement of near-UV light in cataract development suggests that potential anti-cataract drugs may display unusual spectroscopic properties. As bendazac impedes certain effects associated with lens opacification, we have characterized the singlet and triplet states of bendazac and its analog, benzydamine, by fluorescence and phosphorescence methods. These compounds have much shorter triplet state lifetimes compared to the triplet state lifetimes observed in proteins. Our results raise the possibility that the photoprotective action of these compounds may result from their ability to dissipate energy through the triplet state. We propose alternative modes for the photoprotective actions of these compounds.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1996

Stark effect experiments in cytochrome c-type proteins: structural hierarchies.

We performed hole-burning Stark effect experiments on cytochrome c in which the iron of the herne was either removed or replaced by Zn. According to the experiments, the free-base compound has an effective inversion center, even in the protein. The Zn compound, on the other hand, shows quite peculiar features: in the low-frequency range of the inhomogeneous band, it definitely has a dipole moment, as indicated by a splitting of the hole in the external field. However, in the maximum of the inhomogeneous band, a severe charge redistribution occurs, as the experiments show. In addition to the Stark experiments, we performed calculations of the electrostatic fields at the pyrrole rings and at the metal site of the heme group. We interpret our findings with a model based on structural hierarchies: the protein can exist in a few subconformations, which can be distinguished through the structure of the heme pocket. The different pocket structures support different structures of the chromophore, which, in turn, can be distinguished through their behavior in an external field. These distinct structures, in turn, correspond to a rather broad distribution of protein structures, which leave, however, the pocket structure largely unchanged. These structures show up in inhomogeneous broadening.

Animals↗

PubMed1996

Hydrogen atoms are produced when tryptophan within a protein is irradiated with ultraviolet light.

The UV photolysis of the aromatic amino acid, tryptophan (Trp), in the Ca(2+)-binding protein, cod parvalbumin, type III, was studied using electron paramagnetic resonance (EPR) spectroscopy in the temperature range 4-80 K. For the Ca(2+)-bound protein, irradiation with UV light (250-400 nm) resulted in the generation of atomic hydrogen with a hyperfine splitting of 50.9 mT, whereas in the Ca(2+)-free form, where the Trp is exposed to solvent, the trapped atomic hydrogen was not in evidence. In the same spectra, the radical signal in the g = 2.00 region could be detected. The line shape of the Ca(2+)-bound form is similar to the EPR line shape obtained for Trp in micellar systems. In contrast, the EPR line shape for the Ca(2+)-free form is essentially featureless up to 80 K. The EPR spectra of the photoproducts of Trp and the nature of the photoreactions are therefore sensitive to the environment of Trp within the protein.

Animals↗