Denis L. Rousseau

The Layered Structure of Human Mitochondrial DNA Nucleoids

Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.

Resonance Raman scattering of light from a diatomic molecule

Resonance Raman scattering from a homonuclear diatomic molecule is considered in detail. For convenience, the scattering may be classified into three excitation frequency regions—off‐resonance Raman scattering for incident energies well away from resonance with any allowed transitions, discrete resonance Raman scattering for excitation near or in resonance with discrete transitions, and continuum resonance Raman scattering for excitation resonant with continuum transitions, e.g., excitation above a dissociation limit or into a repulsive electronic state. It is shown that the many differences in scattering properties in these three excitation frequency regions may be accounted for by expressions derived from simple perturbation theory. Scattering experiments from molecular iodine are presented which test and verify the general scattering theories. Spectral measurements, time decay measurements, and pressure broadening measurements were made on I2 in the discrete resonance Raman scattering region; and spect...

Domain Swapping in Inducible Nitric-oxide Synthase ELECTRON TRANSFER OCCURS BETWEEN FLAVIN AND HEME GROUPS LOCATED ON ADJACENT SUBUNITS IN THE DIMER

Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and l-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis froml-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 7309–7312). Here, we characterize a pair of heterodimers that contain an l-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented l-Arg binding to one oxygenase domain in each heterodimer but did not affect thel-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-l-Arg interaction confirmed that E371A only prevents l-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.

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 μM, saturation at ~100 μM). 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 ~400 μM, saturation at ~800 μM), 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 μM, 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.

A Cooperative Oxygen Binding Hemoglobin from Mycobacterium tuberculosis STABILIZATION OF HEME LIGANDS BY A DISTAL TYROSINE RESIDUE

The homodimeric hemoglobin (HbN) fromMycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O2, CO, and OH− derivatives of the wild type protein and the B10 Tyr → Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.

Chlamydomonas Chloroplast Ferrous Hemoglobin HEME POCKET STRUCTURE AND REACTIONS WITH LIGANDS

We report the optical and resonance Raman spectral characterization of ferrous recombinantChlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>107 μm −1 s−1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s−1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm−1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.

Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis.

ABSTRACT In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5°C). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5°C, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5°C that is phenotypically indistinguishable from p53-mediated G1arrest at the permissive temperature (32°C). S100B-REF cells proceeding from G1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis.

Solid‐phase epitaxy of implanted silicon by cw Ar ion laser irradiation

Arsenic‐ and antimony‐implanted silicon wafers have been annealed by a cw Ar ion laser. Glancing‐angle Rutherford backscattering and transmission electron and optical microscopy measurements indicate that the mechanism for recrystallization is one of thermal solid‐phase regrowth from the underlying crystalline‐amorphous interface. No implant redistribution is observed.

Pentacoordinate Hemin Derivatives in Sodium Dodecyl Sulfate Micelles: Model Systems for the Assignment of the Fifth Ligand in Ferric Heme Proteins

Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.

Folding intermediates in cytochrome C

Folding of cytochrome c from its low pH guanidine hydrochloride (Gdn-HCl) denatured state revealed a new intermediate, a five-coordinate high spin species with a water molecule coordinated to the heme. Incorporation of this five-coordinated intermediate into the previously reported ligand exchange model can quantitatively account for the observed folding kinetics. In this new model, unfolded cytochrome c is converted to its native structure through an obligatory folding intermediate, the histidine-water coordination state, whereas the five-coordinate state and a bis-histidine state are off-pathway intermediates. When the concentration of Gdn-HCl in the refolding solution was increased, an acceleration of the conversion from the bis-histidine coordinated state to the histidine-water coordinated state was observed, demonstrating that the reaction requires unfolding of the mis-organized polypeptide structure associated with the bis-histidine state.