Biomedical subjects

D L Rousseau

Publications and source records attributed to D L Rousseau.

At least 73 records · Page 4Linked to original sources

PubMed1994

Heme coordination of NO in NO synthase.

A current question in nitric oxide (NO) biology is whether NO can act as a feedback inhibitor of NO synthase (NOS). We have approached this problem by examining the interaction of NO with neuronal NOS by optical absorption and resonance Raman scattering spectroscopies. Under an inert atmosphere NO coordinated to the heme iron in both the oxidized and reduced forms of NOS. The Soret and visible optical absorption transitions are detected at 436 and at 567 nm, respectively, in the Fe(2+)-NO heme complex and at 440 nm and at 549 and 580 nm, respectively, in the Fe(3+)-NO heme complex. In the resonance Raman spectrum of the ferrous complex the Fe-NO stretching mode is located at 549 cm-1 in the presence of L-arginine and at 536 cm-1 in the absence of L-arginine, whereas in the ferric enzyme the mode is located at 540 cm-1 (in the absence of L-arginine). The interaction between bound L-arginine and the NO indicates that L-arginine binds directly over the heme just as do the substrates in cytochrome P-450s. In the absence of L-arginine, NO readily oxidized the ferrous heme iron. The oxidation was prevented by the presence of bound L-arginine and enabled NOS to form a stable ferrous NO complex. Under oxygen-limited conditions, NO generated by neuronal NOS coordinated to its heme iron and formed a spectrally detectable ferrous-NO complex. Taken together, our results show that NO can bind to both ferric and ferrous NOS and may inhibit NO synthesis through its binding to the heme iron during catalysis.

Amino Acid Oxidoreductases↗

PubMed1994

Structural characterization of oxidized dimeric Scapharca inaequivalvis hemoglobin by resonance Raman spectroscopy.

Resonance Raman spectra of the ferric homodimeric hemoglobin from Scapharca inaequivalvis have been measured over the pH range 5.8-8.3 in buffers of ionic strengths 0.01 and 0.1 M to determine the spin and coordination state of the iron atom. Three species contribute to the spectra: a low spin hexacoordinate, a high spin pentacoordinate, and a high spin hexacoordinate component. Optical absorption and EPR spectra measured under the same conditions allowed the identification of the ligands in the sixth coordination position, namely the distal histidine in the low spin derivative and a water molecule in the high spin one. The relative concentrations of these three species depend on pH in an unusual way. Thus, the aquomet derivative is present over the whole pH range, albeit in small amounts as most of the hemoglobin converts to the low spin hemichrome at acid pH values and to the pentacoordinate derivative at neutral and slightly alkaline ones. The formation of a pentacoordinate heme as the pH is increased has not been reported previously for other myoglobins and hemoglobins. Low ionic strength and high protein concentration favor the formation of the high spin pentacoordinate species, while at high ionic strength and low protein concentration the low spin hexacoordinate species prevails. Ionization of the iron-bound water molecule occurs at pH > or = 9.3; accordingly, signals from the hydroxyl derivative were not observed in the Raman spectra over the pH range studied.

Animals↗

PubMed1994

Coordination and spin state equilibria as a function of pH, ionic strength, and protein concentration in oxidized dimeric Scapharca inaequivalvis hemoglobin.

The oxidized homodimeric Scapharca inaequivalvis hemoglobin undergoes changes in coordination and spin state as a function of pH, ionic strength, and protein concentration which have been monitored by optical absorption spectroscopy. Three species contribute to the spectra between pH 5.8 and 8.7: (i) a hexacoordinate high spin aquomet derivative, whose concentration is essentially constant over the whole pH range analyzed; (ii) a pentacoordinate high spin component which prevails at alkaline pH values, and (iii) a hexacoordinate low spin hemichrome, which is formed at acid pH. The contribution of each of the components to the observed spectra was calculated with the singular value decomposition procedure and has been described quantitatively in terms of a linkage scheme which accounts for the change in heme coordination and for the observation that the high spin to low spin transition entails dissociation into monomers. An important feature of the linkage scheme is the cooperative binding of protons to aquomet dimers. Stopped flow experiments to study the kinetics indicate that dissociation into monomers is the rate-limiting process. The unusually strong tendency of oxidized HbI to loose the heme-bound water molecule is discussed in terms of strain in the iron-proximal histidine bond.

Animals↗

PubMed1994

Heme-heme oxygenase complex: structure and properties of the catalytic site from resonance Raman scattering.

The resonance Raman spectra of ferric and ferrous forms of the heme-heme oxygenase (HO) complex (isoform 1) clarify several structural features of the catalytic active site. Isotopic substitution studies of the central iron atom of the heme demonstrate that the line at 218 cm-1 in the ferrous ligand-free form of the complex originates from the iron-histidine stretching mode. The presence of a Raman line at this frequency confirms that the fifth ligand coordinating to the heme is a neutral imidazole from a histidine residue. The modes associated with CO in the carboxy derivative of the ferrous enzyme complex have typical frequencies of histidine-bound heme proteins such as myoglobin. In the ferric form of the complex, at alkaline pH, hydroxide is identified as the bound exogenous ligand, and at neutral pH we infer that water is bound. Thus, the coordination of the heme-HO complex is the same as that in myoglobin. However, in a comparison of the low-frequency vibrational modes in the resonance Raman spectrum of the heme-HO complex to those of myoglobin, the spectra are found to be very different, indicating that the interactions between the heme and its amino acid pocket in these two proteins are quite different. The neutral imidazole may play several important roles in the physiological function of the heme-HO complex.

Animals↗

PubMed1994

Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation.

Heme oxygenase, a central monooxygenase enzyme of the heme catabolism and the associated generation of carbon monoxide, forms a 1:1 stoichiometric complex with iron protoporphyrin IX, which is a prosthetic active center and at the same time the substrate of the enzyme. By using EPR, resonance Raman, and optical absorption spectroscopic techniques, we have determined the axial ligand coordination of the enzyme-heme complex. The ferric heme iron in the heme-enzyme complex at neutral pH is six-coordinate high spin, while at alkaline pH (pKa 7.6), the complex becomes low spin. Spectra of ferrous forms of the complex indicate that histidine serves as the iron proximal axial ligand and that the residue is in its neutral imidazole rather than its imidazolate protonation state. Thus, the active site of the heme-heme oxygenase complex has a myoglobin-like structure rather than an active site similar to the large cytochrome P-450 class of monooxygenases. As a consequence, the activated form of the heme-heme oxygenase complex, a peroxo intermediate, is different from that of the cytochrome P-450 monooxygenases, in which the activated form is an oxo intermediate. The overall catalytic mechanism is probably more closely related to that of other monooxygenases with myoglobin-like active sites, such as secondary amine monooxygenase.

Animals↗

PubMed1993

Heme coordination and structure of the catalytic site in nitric oxide synthase.

Nitric oxide (NO), recently found to play many physiological roles, is generated by the catalysis of L-arginine and O2 to L-citrulline and NO by nitric oxide synthases (NOSs). Resonance Raman spectra from the heme of resting, reduced, and CO-bound forms of rat brain NOS firmly establish that the enzyme belongs to the P-450 class of enzymes. The electron density marker line (V4) in the Raman spectrum of ligand-free ferrous NOS has a low frequency (1347 cm-1), indicating a thiolate axial ligand on the heme. The assignment of a thiolate axial ligand is confirmed in the CO-bound form of the enzyme by the frequency of the Fe-C-O bending mode at 562 cm-1. The heme is resting NOS is five-coordinate high spin and thereby differs from the resting state of most substrate-free P-450s, which are predominantly six-coordinate low spin. The frequency of the Fe-CO stretching mode in the CO-bound enzyme at 491 cm-1, identified by isotope substitution, in higher than that in substrate-free P-450s. Thus, in the ferric and the CO-bound forms of the enzyme, the sixth-ligand binding site on the heme is restricted by steric or hydrophobic interactions. In addition, the Fe-CO stretching mode is broad (30 cm-1) and may be resolved into two overlapping lines of equal intensity, indicating that the heme domains can adopt two distinct conformations.

Amino Acid Oxidoreductases↗

PubMed1993

Preparation and characterization of a bifunctionally spin-labeled mutant of murine epidermal growth factor for saturation-transfer electron paramagnetic resonance studies of the growth factor/receptor complex.

In this report we describe the production of a [Lys3,Tyr22] murine epidermal growth factor (mEGF) mutant for spin-labeling with bis(sulfo-N-succinimidyl)-[15N,2H16]doxyl-2-spiro-4'-pimelate ([15N,2H16]BSSDP) in order to study the rotational dynamics of the EGF/EGF receptor complex by saturation-transfer electron paramagnetic resonance (ST-EPR). Previous results [Faulkner-O'Brien et al. (1991) Biochemistry 30,8976-8985] indicated that the reaction of [15N,2H16]BSSDP with wild-type mEGF did not yield a product useful for ST-EPR studies of the EGF/EGF receptor complex because the major product, in which [15N,2H16]BSSDP was attached only at the amino terminus of mEGF, lacked rigid motional coupling of the spin probe to the protein, and the more tightly coupled bidentate product was unstable. Using oligonucleotide-mediated site-directed mutagenesis of a synthetic gene for mEGF, we replaced Tyr3 with Lys and His22 with Tyr in wild-type mEGF to produce a mutant mEGF suitable for [15N,2H16]BSSDP labeling. The [Lys3,Tyr22] mEGF was expressed in Escherichia coli HB101 transformed with a pIN-III-ompA3-[Lys3,Tyr22] mEGF plasmid and was purified from the bacterial periplasm using a simple two step purification method. The [15N,2H16]BSSDP reacted with [Lys3,Tyr22]mEGF in high yield, and EPR analysis of the major product revealed tight motional coupling between the spin probe and the protein. Biological activity, as assessed by stimulation of EGF receptor autophosphorylation and dimerization, was not affected by either the mutations or the addition of the spin label. The [15N,2H16]BSSDP-modified [Lys3,Tyr22] mEGF was shown to be equipotent with mEGF in EGF receptor competition binding assays using A431 cells; in EPR studies, mEGF also was shown to specifically block [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF binding to the EGF receptor in A431 membrane vesicles. Using the [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF, we now report the first measurement of the rotational dynamics of the EGF/EGF receptor complex in A431 membrane vesicles by ST-EPR.

Amino Acid Sequence↗

PubMed1993

Protein-heme interactions in hemoglobin from the mollusc Scapharca inaequivalvis: evidence from resonance Raman scattering.

Resonance Raman spectra of the Scapharca inaequivalvis homodimeric hemoglobin (HbI) have been measured for the ligand-bound and ligand-free ferrous forms of the protein. In the deoxy derivative, the iron-histidine (Fe-His) stretching mode, proposed as a marker of the oxygen affinity and a conduit linking the hemes to the subunit interface, gives rise to a Raman peak centered at 203 cm-1, an unusually low frequency compared to that reported for other hemoglobins and myoglobins. In the CO-bound derivative, three isotope-sensitive lines at 517, 583, and 1945 cm-1 have been assigned to the Fe-CO stretching, Fe-C-O bending, and C-O stretching modes, respectively. From the frequencies of these modes and from their relative intensities, the Fe-C-O geometry appears to be tilted from axial coordination and shows a bending angle which has been estimated to be about 171 +/- 5 degrees. For the oxygen derivative, only one isotope-sensitive peak has been detected at 570 cm-1, in line with the values found for myoglobin and other hemoglobins. Resonance Raman spectra of HbI modified with p-(chloromercuri)benzoate (PMB) at Cys92 have been measured in parallel with those of the native protein. Despite the large increase in oxygen affinity produced by the PMB modification, the frequency of the Fe-His stretching mode is unshifted in the deoxy derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Heme-heme interactions in a homodimeric cooperative hemoglobin. Evidence from transient Raman scattering.

The comparison of the resonance Raman spectrum of the deoxy dimeric hemoglobin (HbI) from the Arcid clam, Scapharca inaequivalvis, to its CO photoproduct at 10 ns reveals several significant differences in the low frequency vibrational modes including those involving motions of the peripheral substituents on the heme such as the propionates. This finding reflects the involvement of the propionates in a hydrogen bonding network which connects the two heme groups across the subunit interface and is sensitive to ligand binding. A frequency difference in the iron-histidine stretching mode of 6 cm-1 in this invertebrate hemoglobin is substantially smaller than that detected in tetrameric vertebrate hemoglobins under similar conditions. Time evolution studies of the vibrational modes from the photoproduct demonstrate that the transient form relaxes to the deoxy structure concertedly with a half-life of 1 microseconds, the time scale in which tertiary relaxations of tetrameric hemoglobins occur. These data establish that the tertiary and quaternary structural changes take place on the same time scale and confirm a mechanism of cooperativity involving direct interaction between the two heme groups through the peripheral substituents. This direct structural communication provides a very tight linkage between the heme groups prohibiting modulation of the oxygen affinity by factors in the physiological milieu.

Animals↗

PubMed1993

Proton translocation in cytochrome c oxidase: redox linkage through proximal ligand exchange on cytochrome a3.

An analysis of resonance Raman scattering data from CO-bound cytochrome c oxidase and from the photodissociated enzyme indicates that histidine may not be coordinated to the iron atom of cytochrome a3 in the CO-bound form of the enzyme. Instead, the data suggest that either a water molecule or a different amino acid residue occupies the proximal ligand position. From these data, it is postulated that ligand exchange on cytochrome a3 can occur under physiological conditions. Studies of mutant hemoglobins have demonstrated that tyrosinate binds preferentially to histidine in the ferric forms of the proteins. In cytochrome c oxidase tyrosine residues are located near the histidine residues recently implicated in coordination to cytochrome a3 (Shapleigh et al., 1992; Hosler et al., this volume). Expanding on these concepts, we propose a model for proton translocation at the O2-binding site based on proximal ligand exchange between tyrosine and histidine on cytochrome a3. The pumping steps take place at the level of the peroxy intermediate and at the level of the ferryl intermediate in the catalytic cycle and are thereby consistent with the recent results of Wilkstrom (1989) who found that proton pumping occurs only at these two steps. It is shown that the model may be readily extended to account for the pumping of two protons at each of the steps.

Binding Sites↗

PubMed1993

Metastable CO binding sites in the photoproduct of a novel cooperative dimeric hemoglobin.

The infrared absorption spectrum of the CO-photoproduct from Scapharca inaequivalvis hemoglobin (Hbl) at 10 K yields only a single line in the "B" state region at 2132 cm-1. This is the same frequency as the B1 line observed in photodissociated vertebrate HbCO and MbCO. No evidence was found for the B2 line detected in vertebrate hemoglobins and myoglobin in the 2118-2120 cm-1 region. These data demonstrate that the protein does not have the same conformationally accessible ligand-binding sites as do vertebrate hemoglobins and myoglobins. The absence of the B2 line indicates that only a single weak site is accessible to the photolyzed CO molecule. These results are in accord with geminate rebinding experiments and ligand escape pathway calculations which have shown that the distal properties of Hbl are distinct from those of tetrameric hemoglobins and vertebrate myoglobins.

Animals↗

PubMed1991

Vibrational structure of the formyl group on heme a. Implications on the properties of cytochrome c oxidase.

Resonance Raman spectra have been recorded for heme a derivatives in which the oxygen atom of the formyl group has been isotopically labeled and for Schiff base derivatives of heme a in which the Schiff base nitrogen has been isotopically labeled. The 14N-15N isotope shift in the C = N stretching mode of the Schiff base is close to the theoretically predicted shift for an isolated C = N group for both the ferric and ferrous oxidation states and in both aqueous and nonaqueous solutions. In contrast, the 16O-18O isotope shift of the C = O stretching mode of the formyl group is significantly smaller than that predicted for an isolated C = O group and is also dependent on whether the environment is aqueous or nonaqueous. This differences between the theoretically predicted shifts and the observed shifts are attributed to coupling of the C = O stretching mode to as yet unidentified modes of the heme. The complex behavior of the C = O stretching vibration precludes the possibility of making simple interpretations of frequency shifts of this mode in cytochrome c oxidase.

Electron Transport Complex IV↗

PubMed1990

Ferryl and hydroxy intermediates in the reaction of oxygen with reduced cytochrome c oxidase.

Cytochrome c oxidase catalyses the 4-electron reduction of dioxygen to water and translocates protons vectorially across the inner mitochondrial membrane. Proposed reaction pathways for the catalytic cycle of the O2 reduction are difficult to verify without knowing the structures of the intermediates, but we now have such information for the catalytic intermediates in the first steps of the reaction of O2 with cytochrome c oxidase from resonance Raman spectroscopy, a technique that enables iron-ligand stretching modes to be identified. Here we report on two more key intermediates: a ferryl-oxo (Fe4 = O2-) and a ferric-hydroxy (Fe3+--OH-) intermediate at the level of 3- and 4-electron reduction, respectively. We identified these intermediates by their characteristic iron-oxygen stretching frequencies (786 cm-1 for Fe4+ = O2-, and 450 cm-1 for Fe3+ -- OH-) and oxygen and deuterium isotope shifts. The oxo atom in the ferryl intermediate is hydrogen-bonded and the iron-oxygen bond in the hydroxy intermediate is anomalously weak. With the identification of the primary, ferryl and hydroxy intermediates, the predominant structures at almost all stages of O2 reduction are now known and the catalytic pathway can be described with more certainty.

Deuterium↗

PubMed1990

Primary intermediate in the reaction of mixed-valence cytochrome c oxidase with oxygen.

The reaction of dioxygen with mixed-valence cytochrome c oxidase was followed in a rapid-mixing continuous-flow apparatus. The optical absorption difference spectrum and a kinetic analysis confirm the presence of the primary oxygen intermediate in the 0-100-microseconds time window. The resonance Raman spectrum of the iron-dioxygen stretching mode (568 cm-1) supplies evidence that the degree of electron transfer from the iron atom to the dioxygen is similar to that in oxy complexes of other heme proteins. Thus, the Fe-O2 bond does not display any unique structural features that could account for the rapid reduction of dioxygen to water. Furthermore, the frequency of the iron-dioxygen stretching mode is the same as that of the primary intermediate in the fully reduced enzyme, indicating that the oxidation state of cytochrome a plays no role in controlling the initial properties of the oxygen binding site.

Chemical Phenomena↗

PubMed1990

Metastable intermediates in myoglobin at low pH.

Resonance Raman and optical absorption spectra of ligand-free (deoxy) myoglobin and CO-bound myoglobin (MbCO) at pH 2.6 have been measured by using continuous-flow/rapid-mixing techniques. The spectra of deoxy myoglobin at low pH within 6 ms of the pH drop demonstrate that the iron-histidine bond has been ruptured but that the heme is still five-coordinate. Comparison with data from model complexes indicates that a weak-field ligand, such as a water molecule, is coordinated at the fifth position. The Raman spectrum of MbCO at low pH has an Fe-CO stretching mode that is characteristic of a six-coordinate heme with an unhindered Fe-CO moiety. Immediately following the pH drop in this case, there is no indication that the iron-proximal histidine bond is broken. Three different structural changes are detected at low pH: (i) the iron-proximal histidine (F8) bond in ligand-free myoglobin is broken and replaced by a weak-field ligand, (ii) the distal pocket in MbCO is opened, and (iii) protein constraints on the heme group in MbCO are relaxed. Previous conclusions that the kinetics of CO-binding in hemoproteins at low pH is modified by rupturing the iron-proximal histidine bond are supported by these new results which, however, demand a more complete reevaluation of the phenomenon.

Animals↗

PubMed1990

Cytochrome c oxidase: decay of the primary oxygen intermediate involves direct electron transfer from cytochrome a.

The decay of the primary intermediate generated in the reaction of oxygen with cytochrome c oxidase is nearly one order of magnitude faster in the fully reduced form of the enzyme than it is in the mixed valence form. To account for this observation, we propose a model describing the early molecular events in the reaction. In this model the decay of the primary Fe-O2 intermediate in the fully reduced enzyme is a consequence of direct electron transfer from cytochrome a. To test the model we measured the time dependence of the oxidation of cytochrome a by monitoring the resonance Raman scattering intensity of its vibrational modes. A rapid oxidation of cytochrome a was detected that quantitatively agrees with the model. These results indicate that the mechanism of oxygen reduction and proposed frameworks for proton translocation must be reexamined.

Animals↗

PubMed1990

Primary intermediate in the reaction of oxygen with fully reduced cytochrome c oxidase.

The primary intermediate in the reaction of oxygen with cytochrome c oxidase was generated by photodissociating carbon monoxide in a continuous flow rapid mixing apparatus. The presence of the primary intermediate was confirmed by a comparison of the iron-dioxygen stretching frequency with that obtained in the reaction of oxygen with the mixed-valence enzyme. For both of these preparations, the Fe-O2 stretching mode is detected at 568 cm-1, the same frequency as that found in oxyhemoglobin and oxymyoglobin. These data illustrate that the primary intermediate may be generated and detected at room temperature in the fully reduced enzyme and that the oxidation state of cytochrome a does not affect the structure of the iron-dioxygen complex. By following the changes in the intensity of the Fe-O2 stretching mode in the resonance Raman spectrum as a function of time, the first-order rate constant for the decay of the primary intermediate was found to be 3.5 x 10(4) s-1 (t1/2 = 20 microseconds).

Carbon Monoxide↗

PubMed1989

Spin equilibrium in human methemoglobin: effects of inositol hexaphosphate and bezafibrate as measured by resonance Raman spectroscopy.

The spin equilibria of several derivatives of human methemoglobin were probed by resonance Raman scattering. The intensity of lines in the Raman spectrum gives a measure of the high-spin (S = 5/2) to low-spin (S = 1/2) ratio which agrees well with the spin equilibria determined from direct magnetic susceptibility measurements. The addition of bezafibrate (BZF) to methemoglobin in the absence of organic phosphate, IHP, has very little effect on the spin equilibrium, whereas in the presence of IHP it augments the change in spin significantly. When both IHP and BZF are added to the mixed-spin derivatives (H2O, SCN-, OCN-, and NO2-) of human methemoglobin, the spin equilibrium is shifted toward higher spin by about 700 cal/mol, similar to the spin change detected in derivatives of carp methemoglobin upon addition of IHP alone. These data support a general mechanism for the allosteric transition in which a constant fraction of the cooperative energy (approximately 20%) is detected at the heme of the ferric ligand-bound forms.

Animals↗