Biomedical subjects

Pengchi Deng

Publications and source records attributed to Pengchi Deng.

9 recordsLinked to original sources

PubMed2006

Spectroscopic study of substrate binding to the carbonmonoxy form of dehaloperoxidase from Amphitrite ornata.

Dehaloperoxidase (DHP) is a globular heme enzyme found in the marine worm Amphitrite ornata that can catalyze the dehalogenation of halophenols to the corresponding quinones by using hydrogen peroxide as a cosubstrate. Its three-dimensional fold is surprisingly similar to that of the oxygen storage protein myoglobin (Mb). A key structural feature common to both DHP and Mb is the existence of multiple conformations of the distal histidine. In DHP, the conformational flexibility may be involved in promotion of substrate and cosubstrate entry and exit. Here we have explored the dynamics of substrate binding in DHP using Fourier transform infrared spectroscopy and flash photolysis. A number of discrete conformations at the active site were identified from the appearance of multiple CO absorbance bands in the infrared region of the spectrum. Upon photolysis at cryogenic temperatures, the CO molecules are trapped at docking sites within the protein matrix, as inferred from the appearance of several photoproduct bands characteristic of each site. Substrate binding stabilizes the protein by approximately 20 kJ/mol. The low yield of substrate-bound DHP at ambient temperature points toward a steric inhibition of substrate binding by carbon monoxide.

Animals↗

PubMed2004

Probing electric fields in protein cavities by using the vibrational stark effect of carbon monoxide.

To determine the magnitude and direction of the internal electric field in the Xe4 cavity of myoglobin mutant L29W-S108L, we have studied the vibrational Stark effect of carbon monoxide (CO) using infrared spectroscopy at cryogenic temperatures. CO was photodissociated from the heme iron and deposited selectively in Xe4. Its infrared spectrum exhibits Stark splitting into two bands associated with CO in opposite orientations. Two different photoproduct states can be distinguished, C' and C'', with markedly different properties. For C', characteristic temperature-dependent changes of the area, shift, and width were analyzed, based on a dynamic model in which the CO performs fast librations within a double-well model potential. For the barrier between the wells, a height of approximately 1.8 kJ/mol was obtained, in which the CO performs oscillations at an angular frequency of approximately 25 cm(-1). The magnitude of the electric field in the C' conformation was determined as 11.1 MV/cm; it is tilted by an angle of 29 degrees to the symmetry axis of the potential. Above 140 K, a protein relaxation leads to a significantly altered photoproduct, C'', with a smaller Stark splitting and a more confining potential (barrier >4 kJ/mol) governing the CO librations.

Animals↗

PubMed2004

Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin.

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 = 200-600 s(-1)) and moderate O2 affinity (KO2) approximately 1 microm(-1)) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 degrees C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 --> Val CerHbO2 at 1.70 A resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 A). The dramatic functional and spectral effects of the Thr-E11 --> Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm(-1) decrease in nuCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.

Animals↗

PubMed2003

Structural dynamics of myoglobin: spectroscopic and structural characterization of ligand docking sites in myoglobin mutant L29W.

We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, A(I) and A(II), with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, A., et al. (2000) Nature 404, 205-208]. The detailed knowledge of the interplay between protein structure, protein dynamics, and ligand migration at cryogenic temperatures allowed us to develop a dynamic model that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.

Amino Acid Substitution↗

PubMed2003

Structural dynamics of myoglobin: effect of internal cavities on ligand migration and binding.

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.

Amino Acid Substitution↗

PubMed2003

Structural dynamics of myoglobin: ligand migration and binding in valine 68 mutants.

We have combined Fourier transform infrared/temperature derivative (FTIR-TDS) spectroscopy at cryogenic temperatures and flash photolysis at ambient temperature to examine the effects of polar and bulky amino acid replacements of the highly conserved distal valine 68 in sperm whale myoglobin. In FTIR-TDS experiments, the CO ligand can serve as an internal voltmeter that monitors the local electrostatic field not only at the active site but also at intermediate ligand docking sites. Mutations of residue 68 alter size, shape, and electric field of the distal pocket, especially in the vicinity of the primary docking site (state B). As a consequence, the infrared bands associated with the ligand at site B are shifted. The effect is most pronounced in mutants with large aromatic side chains. Polar side chains (threonine or serine) have only little effect on the peak frequencies. Ligands that migrate toward more remote sites C and D give rise to IR bands with altered frequencies. TDS experiments separate the photoproducts according to their recombination temperatures. The rates and extent of ligand migration among internal cavities at cryogenic temperatures can be used to interpret geminate and bimolecular O2 and CO recombination at room temperature. The kinetics of geminate recombination can be explained by steric arguments alone, whereas both the polarity and size of the position 68 side chain play major roles in regulating bimolecular ligand binding from the solvent.

Amino Acid Substitution↗

PubMed2003

Ligand dynamics in a protein internal cavity.

We have studied the temperature dependence of the IR stretch bands of carbon monoxide (CO) in the Xe 4 internal cavity of myoglobin mutant L29W-S108L at cryogenic temperatures. Pronounced changes of band areas and positions were analyzed quantitatively by using a simple dynamic model in which CO rotation in the cavity is constrained by a static potential. The librational dynamics of the CO causes a decrease of the total spectral area. A strong local electric field splits the CO stretch absorption into a doublet, indicating that CO can assume opposite orientations in the cavity. With increasing temperature, the two peaks approach each other, because the average angle of the CO with respect to the electric field increases. A combined classical and quantum-mechanical analysis precisely reproduces the observed temperature dependencies of both spectral area and peak shifts. It yields the height of the energy barrier between the two wells associated with opposite CO orientations, V0 approximately 2 kJ/mol, and the frequency of oscillation within a well, omega approximately 25 cm(-1). The electric field in the protein cavity was estimated as 10 MV/cm.

Animals↗

PubMed2002

Ligand binding and protein dynamics in neuroglobin.

Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins. It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown. Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions. To bind exogenous ligands like O(2) and CO, the His E7 endogenous ligand is displaced from the sixth coordination. By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3-353 K). Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO. Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers. The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K. Flash photolysis near physiological temperature (275-353 K) exhibits four sequential kinetic features: (i) geminate rebinding (t < 1 micros); (ii) extremely fast bimolecular exogenous ligand binding (10 micros < t < 1 ms) with a nontrivial temperature dependence; (iii) endogenous ligand binding (100 micros < t < 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (iv) displacement of the endogenous by the exogenous ligand (10 ms < t < 10 ks). All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales.

Animals↗

PubMed2002

The effect of ligand dynamics on heme electronic transition band III in myoglobin.

Band III is a near-infrared electronic transition at ~13,000 cm(-1) in heme proteins that has been studied extensively as a marker of protein conformational relaxation after photodissociation of the heme-bound ligand. To examine the influence of the heme pocket structure and ligand dynamics on band III, we have studied carbon monoxide recombination in a variety of myoglobin mutants after photolysis at 3 K using Fourier transform infrared temperature-derivative spectroscopy with monitoring in three spectral ranges, (1) band III, the mid-infrared region of (2) the heme-bound CO, and (3) the photodissociated CO. Here we present data on mutant myoglobins V68F and L29W, which both exhibit pronounced ligand movements at low temperature. From spectral and kinetic analyses in the mid-infrared, a small number of photoproduct populations can be distinguished, differing in their distal heme pocket conformations and/or CO locations. We have decomposed band III into its individual photoproduct contributions. Each photoproduct state exhibits a different "kinetic hole-burning" (KHB) effect, a coupling of the activation enthalpy for rebinding to the position of band III. The analysis reveals that the heme pocket structure and the photodissociated CO markedly affect the band III transition. A strong kinetic hole-burning effect results only when the CO ligand resides in the docking site on top of the heme group. Migration of CO away from the heme group leads to an overall blue shift of band III. Consequently, band III can be used as a sensitive tool to study ligand dynamics after photodissociation in heme proteins.

Animals↗