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D L Rousseau

Publications and source records attributed to D L Rousseau.

At least 55 records · Page 3Linked to original sources
PubMed1997

The unique heme-heme interactions of the homodimeric Scapharca inaequivalvis hemoglobin as probed in the protein reconstituted with unnatural 2,4 heme derivatives.

In the homodimeric hemoglobin from Scapharca, HbI, functional communication between the two heme groups is based on their direct structural linkage across the subunit interface through the heme propionates. The heme-protein interactions have been altered in deutero- and meso-HbI by substituting the vinyl groups at positions 2 and 4 of protoheme with hydrogen and ethyl groups, respectively. In meso-HbI the introduction of the ethyl groups in the heme pocket induces significant alterations in the conformation of the heme peripheral substituents, including the propionates, and in the structure of bound CO, as revealed by the resonance Raman spectra. The functional counterpart of these structural changes is the loss of cooperativity in carbon monoxide binding and in the rate of oxygen dissociation. Oxygen pulse and flash photolysis experiments indicate that meso-HbI is locked in the liganded conformation. It is postulated that the ethyl groups, which occupy a larger volume than vinyl ones, impair the ligand-linked movement of the heme relative to its pocket and in turn the expression of cooperativity. In deutero-HbI structural alterations have not been monitored. Functionally, cooperativity in the CO binding kinetics is increased as if hydrogen atoms at positions 2 and 4 permitted more marked movements of the heme than in the native protein.

Animals↗
PubMed1997

Submillisecond protein folding kinetics studied by ultrarapid mixing.

An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent flow created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 micros to 3 ms. To eliminate the heme-ligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The fluorescence decrease is biphasic. There is an unresolved process with tau < 50 micros, followed by a slower, exponential process with tau = 600 micros at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.

Animals↗
PubMed1997

Resonance Raman spectroscopic characterization of alpha-hydroxyheme and verdoheme complexes of heme oxygenase.

Heme oxygenase (HO) is the microsomal enzyme that catalyzes the oxidative degradation of protoheme (iron protoporphyrin IX) and the generation of carbon monoxide. The enzyme converts protoheme into biliverdin through two known heme derivatives, alpha-hydroxyheme and verdoheme. To gain insight into the degradation mechanisms of the two intermediates, the resonance Raman spectra were observed for alpha-hydroxyheme and verdoheme complexes of HO and compared with those of apomyoglobin (apo-Mb) complexes. The ferrous alpha-hydroxyheme complexed with both HO and apo-Mb shows a resonance Raman spectral pattern similar to that of the protoheme complexes. On the contrary, the ferric alpha-hydroxyheme and ferrous verdoheme complexes of HO and apo-Mb show atypical Raman patterns, which are interpreted as the result of the symmetry lowering of the porphyrin-conjugated pi-electron system. The comparison of the resonance Raman spectra of the verdoheme complexed with HO and apo-Mb with those of the five- and six-coordinate model complexes of verdoheme shows that the ferrous forms of the verdoheme-protein complexes are six-coordinate. The Fe-CO and Fe-CN stretching frequencies of ferrous verdoheme compounds are distinct from those of ferrous heme compounds. It is inferred that the positive charge of the verdoheme ring possesses some of the charge density on the iron atom, causing unique characteristics of the iron ligand stretching vibrations and altered ligand binding properties.

Heme↗
PubMed1997

Folding of cytochrome c initiated by submillisecond mixing.

Cytochrome c folding was initiated using a new solution mixer that provides a time window which covers over 90% of the burst phase unresolved by conventional stop-flow measurements. Folding was followed by resonance Raman scattering. Kinetic analysis of the high frequency Raman data indicates that a nascent phase occurs within the mixing dead time of 100 microseconds. A significant fraction of the protein was found to be trapped in a misfolded bis-histidine form during the nascent phase at pH 4.5, thereby preventing the protein from folding rapidly and homogeneously. The nascent phase was followed by a haem-ligand exchange phase that populates the native histidine-methionine coordinated form through a thermodynamically controlled equilibrium.

Animals↗
PubMed1997

Ligand exchange during cytochrome c folding.

Submillisecond folding of cytochrome c reveals that a nascent phase appears within the mixing dead time of 100 microseconds, followed by a ligand exchange reaction during which His 26/33, water and Met 80 are inter-exchanged as haem ligands through a thermodynamically controlled equilibrium. In the ligand exchange phase, the rate of formation of a misfolded histidine-histidine coordinated state (HH) decreases by two orders of magnitude as the pH is reduced from 5.9 to 4.5 due to the protonation of the misligated His 26/33. The activation energy barriers for the transitions from the histidine-water coordinated form (HW) to the histidine-methionine coordinated form and the HH form are 18 and 4 kcal mol-1 respectively, at pH 4.8. The activation energy barrier for protein to escape from the misligated HH to the HW form was measured to be 12 kcal mol-1, demonstrating the kinetic trapping effect of the misligated bis-histidine form. The development of the polypeptide tertiary structure near the haem is concomitant with the coordination of the native haem axial ligand.

Animals↗
PubMed1996

Nitric oxide binding to the heme of neuronal nitric-oxide synthase links its activity to changes in oxygen tension.

Neuronal nitric-oxide synthase (NOS-1) is a hemeprotein that generates NO and citrulline from L-arginine, O2, and NADPH. During catalysis, a majority of NOS-1 binds self-generated NO and converts to a ferrous-NO complex, which causes it to operate at a fraction of its maximum possible activity during the steady state (Abu-Soud, H. M., Wang, J., Rousseau, D. L., Fukuto, J., Ignarro, L. J., and Stuehr, D. J. (1995) J. Biol. Chem. 270, 22997-23006). To examine how NO complex formation affects the O2 response of NOS-1, we measured rates of NO synthesis and NADPH oxidation versus O2 concentration in the presence and absence of L-arginine. In the absence of L-arginine, NOS-1 catalyzed simple O2 reduction, and its heme iron displayed a typical affinity for O2 (estimated KmO2 </= 40 microM, saturation at approximately 100 microM). In the presence of L-arginine, the rates of NO synthesis and NADPH oxidation were proportional to the O2 concentration over a much broader range (estimated KmO2 approximately 400 microM, saturation at approximately 800 microM), indicating that ferrous-NO complex formation altered the O2 response of NOS-1. Stopped-flow experiments revealed that the rate of ferrous-NO complex formation was relatively independent of the O2 concentration between 100 and 700 microM, while the rate of complex breakdown was directly proportional to O2 concentration. We conclude that the O2 sensitivity of the ferrous-NO complex governs the O2 response of NOS-1 and thus its activity during the steady state. This enables NOS-1 to couple its rate of NO synthesis to the O2 concentration throughout the physiologic range.

Calmodulin↗
PubMed1996

Electric-field-induced Schiff-base deprotonation in D85N mutant bacteriorhodopsin.

The application of an external electric field to dry films of Asp-85-->Asn mutant bacteriorhodopsin causes deprotonation of the Schiff base, resulting in a shift of the optical absorption maximum from 600 nm to 400 nm. This is in marked contrast to the case of wild-type bacteriorhodopsin films, in which electric fields produce a red-shifted product whose optical properties are similar to those of the acid-blue form of the protein. This difference is due to the much weaker binding of the Schiff-base proton in the mutant protein, as indicated by its low pK of approximately 9, as compared with the value pK approximately 13 in the wild type. Other bacteriorhodopsins with lowered Schiff-base pK values should also exhibit a field-induced shift in the protonation equilibrium of the Schiff base. We propose mechanisms to account for these observations.

Amino Acid Sequence↗
PubMed1996

Oxygen and one reducing equivalent are both required for the conversion of alpha-hydroxyhemin to verdoheme in heme oxygenase.

Heme oxygenase is a central enzyme of heme degradation and associated carbon monoxide biosynthesis. We have prepared the alpha-hydroxyheme-heme oxygenase complex, which is the first intermediate in the catalytic reaction. The active site structure of the complex was examined by optical absorption, EPR, and resonance Raman spectroscopies. In the ferric form of the enzyme complex, the heme iron is five coordinate high spin and the alpha-hydroxyheme group in the complex assumes a structure of an oxophlorin where the alpha-meso hydroxy group is deprotonated. In the ferrous form, the alpha-hydroxy group is protonated and consequently the prosthetic group assumes a porphyrin structure. The alpha-hydroxyheme group undergoes a redox-linked conversion between a keto and an enol form. The ferric alpha-hydroxyheme reacts with molecular oxygen to form a radical species. Reaction of the radical species with a reducing equivalent yields the verdoheme-heme oxygenase complex. Reaction of the ferrous alpha-hydroxyheme-heme oxygenase complex with oxygen also yields the verdoheme-enzyme complex. We conclude that the catalytic conversion of ferric alpha-hydroxyheme to verdoheme by heme oxygenase requires molecular oxygen and one reducing equivalent.

Animals↗
PubMed1996

Cytochrome c peroxidase complexed with cytochrome c has an unperturbed heme moiety.

Transient resonance Raman, Raman difference, circular dichroism (CD), and optical absorption studies have been carried out on the electrostatic complexes formed by yeast cytochrome c peroxidase (CCP) with horse cytochrome c (Cytc) in low ionic strength solutions. In all the complexes examined [e.g., CCP(II)/Cytc(II), CCP(III)/Cytc(II), CCP(III)/Cytc(III)], the local heme environments of both proteins are largely unperturbed upon complexation. Specifically, CCP preserves a completely pentacoordinate high-spin heme in both its ferric and ferrous forms in CCP/Cytc complexes and uncomplexed mixtures. We found no evidence corroborating the previously reported increase in the low-spin fraction of CCP heme upon complexation with Cytc [Hildebrandt et al. (1992) Biochemistry 31, 2384-2392]. Instead, our Raman data strongly suggest that the H-bonding networks in the distal and proximal pockets of CCP are well maintained in the complexes. On the other hand, CD spectra of CCP(III)/Cytc(III) complexes showed substantial variations (relative to the uncomplexed mixtures) in the far-UV region, reflecting some protein conformational rearrangements. In addition, the spectral data suggest that complexation with Cytc affects the previously observed pH-dependent flexibility of the heme structure of CCP and thus influences the photodynamics of the CCP active site.

Amino Acid Sequence↗
PubMed1995

Reaction of cytochrome bo3 with oxygen: extra redox center(s) are present in the protein.

The reaction of oxygen with cytochrome bo3, a quinol oxidase from Escherichia coli, has been studied by resonance Raman scattering after initiation of the reaction by CO photolysis in a continuous flow apparatus and by directly mixing the enzyme with oxygen. The high-frequency region of the spectrum was monitored to determine the time evolution of the spin, oxidation, and coordination states of heme O and the oxidation state of heme B by using newly established marker lines for each heme. Three phases of the reaction were detected. In phase I, complete in 75 microseconds, O2 reacted with heme O and formed a low-spin ferric or ferryl adduct without significant oxidation of heme B. In phase II, between 75 and 120 microseconds, a small fraction of heme B was oxidized. In phase III, at approximately 1 s, the majority of heme B was oxidized and heme O reverted to a high-spin ferric state. The high rate of oxygen reduction at heme O to the three- or four-electron reduced level, despite a very low rate of heme B oxidation, indicates that there are electron donors active in the enzyme other than the metal centers. Assays of our enzyme preparations rule out a quinol in the tight binding (QH) site as a possible donor but instead suggest electron donation from the protein matrix, such as from tryptophans or tyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Simulation↗
PubMed1995

The interaction of epidermal growth factor with its receptor in A431 cell membranes: a stopped-flow fluorescence anisotropy study.

We describe a quantitative examination of the interaction of epidermal growth factor (EGF) with the EGF receptor using A431 cell membrane vesicles as a receptor source. Using T-format steady-state fluorescence anisotropy detection coupled with stopped-flow mixing, we measured the association and EGF-induced dissociation kinetics of fluorescein 5-isothiocyanate-labeled mEGF (FITC-EGF) with the EGF receptor over a wide range of FITC-EGF concentrations, membrane dilutions, and time scales (milliseconds to minutes). Fluorescence anisotropy-based equilibrium binding titrations were also performed. All studies utilized the same receptor preparation, ligand preparation, and detection system. The entire data surface (approximately 78,000 data points) was simultaneously analyzed using global analysis techniques with a variety of kinetic models. Our analysis identified with a high level of confidence receptor populations with two association rate constants (k(on) = 1.2 x 10(6) M-1 s-1, 7.2 x 10(6) M-1 s-1) and three dissociation rate constants (k(off0 = 0.95 x 10(-2) s-1, 0.13 x 10(-2) s-1, 0.32 x 10(-3) s-1), which reflect the presence of at least two distinct receptor populations in A431 cell membranes. Analysis of the kinetic data was found to be much more sensitive to the presence of multiple receptor populations than was the analysis of the equilibrium binding data.

Animals↗
PubMed1995

Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis.

Neuronal NO synthase (NOS) is a flavin-containing hemeprotein that generates NO from L-arginine, NADPH, and O2. NO has recently been proposed to autoinhibit NOS. We have investigated whether a NOS heme-NO complex forms during aerobic steady-state catalysis. Visible and resonance Raman spectra recorded during steady-state NO synthesis by NOS showed that the majority of enzyme (70-90%) was present as its ferrous-nitrosyl complex. Ferrous-nitrosyl NOS formed only in the coincident presence of NADPH, L-arginine, and O2. Its level remained constant during NO synthesis until the NADPH was exhausted, after which the complex decayed to regenerate ferric resting NOS. Stopped-flow measurements revealed that the buildup of the ferrous-NO complex was rapid (< 2 s) and caused a 10-fold decrease in the rate of NADPH consumption by NOS. Complex formation and decay could occur several times with no adverse affect on its subsequent formation or on NOS catalytic activity. Neither enzyme dilution nor NO scavengers (superoxide and oxyhemoglobin) diminished formation of ferrous-nitrosyl NOS or prevented the catalytic inhibition attributed to its formation. The ferrous-nitrosyl complex also formed in unfractionated cell cytosol containing neuronal NOS upon initiating NO synthesis. We conclude that a majority of neuronal NOS is converted quickly to a catalytically inactive ferrous-nitrosyl complex during NO synthesis independent of the external NO concentration. Thus, NO binding to the NOS heme may be a fundamental feature of catalysis and functions to down-regulate NO synthesis by neuronal NOS.

Aerobiosis↗
PubMed1995

Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site.

Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins. One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000- to 1070-cm-1 region. The other line is found in the 1180- to 1210-cm-1 region and is assigned as a combination between a porphyrin mode, nu 7, and the Fe-CO stretching mode. The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands. The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family. Furthermore, the anharmonicity correlates with the frequency of the C-O stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy. The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties.

Animals↗
PubMed1995

Two conformations of the catalytic site in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides.

Resonance Raman spectra of the carbon monoxy derivative of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides show two distinct Fe-CO stretching modes (519 and 493 cm-1) at room temperature. The frequency of the mode at 519 cm-1 coincides with that of other terminal oxidases at neutral pH. Two C-O stretching modes, one at 1966 cm-1 and one at 1955 cm-1, are also found. The splitting of the C-O stretching mode is consistent with the FTIR spectra of cytochrome c oxidases at cryogenic temperatures in which two different conformations (alpha and beta) of the catalytic site of the enzyme are present. The splitting of both the Fe-CO and C-O stretching modes under our conditions indicates that these two forms of the enzyme are also present at room temperature, and with the additional information on the Fe-CO modes provided here, a structural origin for the two forms may be postulated. The alpha-form has the same general structure of the active site as mammalian oxidase, a structure in which the copper atom that is the part of the Fe-CuB binuclear site interacts strongly with the bound CO. We postulate that the copper atom exerts a strong polar or steric effect on the heme-bound CO, resulting in either compression of the Fe-CO bond or distortion of the Fe-CO moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Tetrahydrobiopterin-deficient nitric oxide synthase has a modified heme environment and forms a cytochrome P-420 analogue.

Optical absorption and resonance Raman spectra of neuronal nitric oxide synthase (b-NOS) isolated in the absence of tetrahydrobiopterin demonstrate that the enzyme preparation is very unstable. This unstable form of the enzyme has properties analogous to those of cytochrome P-420cam, an inactive form of cytochrome P-450cam. Although cysteine is preserved as the proximal ligand in both the ferric and ferrous forms of unstable b-NOS, the lack of tetrahydrobiopterin significantly increases the hexacoordinate low-spin fraction of the heme content, resulting in a loss of the enzymatic activity. Upon the addition of CO, the unstable b-NOS converts from a species exhibiting a Soret absorption maximum at 443 nm, as reported for the CO adducts of stable b-NOS and cytochrome P-450cam, to a species with a Soret maximum at 421 nm. The resonance Raman spectrum of the 421-nm form is the same as those of CO-bound myoglobin at low pH and CO-bound cytochrome P-420cam. The heme in this form of the enzyme is coordinated by a weaker ligand than thiolate; histidine coordination in the CO-bound form of the P-420-like species of NOS is consistent with all of the available data. A similar unstable form of the macrophage (i-NOS) enzyme was also detected. Not only does the heme pocket of NOS have the same coordination as cytochrome P-450 in its stable form, but the partially denatured form has the same properties as cytochrome P-420, the inactive form of cytochrome P-450. Among other possible roles, tetrahydrobiopterin may play a significant role in the stabilization of the active enzyme.

Amino Acid Oxidoreductases↗
PubMed1995

Microsecond generation of oxygen-bound cytochrome c oxidase by rapid solution mixing.

Current understanding of the oxygen reduction and proton translocation processes in cytochrome c oxidase is largely derived from the data obtained by a nonphysiological method for initiating the catalytic reaction: photolyzing carbon monoxide (CO) from the CO-inhibited enzyme in the presence of oxygen (O2). However, considerable evidence suggests that the use of CO introduces artifacts into the reaction mechanism. We have therefore developed a rapid solution mixer with a mixing time of 20 microseconds to study the catalytic reaction by directly mixing the enzyme with O2 without using CO. Unexpectedly, the resonance Raman scattering detected for the first 120 microseconds after the mixing show that the CO influences neither the structure of the primary oxy-intermediate, its rate of decay, nor the rate of oxidation of cytochrome a. This implies that CO has an effect on the later stages of the catalytic process, which may involve the proton translocation steps, and calls for the re-examination of the catalytic process by using the direct mixing method. In addition, these results demonstrate the feasibility of using the rapid mixing device for the study of biological reactions in the microsecond time domain.

Animals↗
PubMed1995

Heme oxygenase-2. Properties of the heme complex of the purified tryptic fragment of recombinant human heme oxygenase-2.

Recombinant human microsomal heme oxygenase-2 was expressed in Escherichia coli. Tryptic digestion of the membrane fraction, in which the wild-type enzyme was localized, yielded a soluble tryptic peptide of 28 kDa, which retained the ability to accept electrons from NADPH-cytochrome P-450 reductase and the enzymatic activity for conversion of heme to biliverdin. The tryptic fragment, when purified to apparent homogeneity, bound one equivalent of heme to form a substrate-enzyme complex that had spectroscopic properties characteristic of heme proteins, such as myoglobin and hemoglobin. Optical absorption, Raman scattering, and EPR studies of the heme-tryptic fragment complex revealed that the ferric heme was six coordinate high spin at neutral pH and six coordinate low spin at alkaline pH, with a pK alpha value of 8.5. EPR and Raman scattering studies indicated that a neutral imidazole of a histidine residue served as the proximal ligand in the heme-heme oxygenase-2 fragment complex. The reaction with hydrogen peroxide converted the heme of the heme oxygenase-2 fragment complex into a verdoheme-like intermediate, while the reaction with m-chloroperbenzoic acid yielded a oxoferryl species. These spectroscopic properties are similar to those obtained for heme oxygenase-1, and thus the catalytic mechanism of heme oxygenase-2 appears to be similar to that of heme oxygenase-1.

Amino Acid Sequence↗