H. Jouve

Ligand diffusion in the catalase from Proteus mirabilis: A molecular dynamics study

The role of the channels and cavities present in the catalase from Proteus mirabilis (PMC) was investigated using molecular dynamics (MD) simulations. The reactant and products of the reaction, H2O2 →1/2 O2 + H2O, catalyzed by the enzyme were allowed to diffuse to and from the active site. Dynamic fluctuations in the structure are found necessary for the opening of the major channel, ideied in the X‐ray model, which allows access to the active site. This channel is the only pathway to the active site observed during the dynamics, and both the products and reactant use it. H2O and O2 are also detected in a cavity defined by the heme and Ser196, which could play an important role during the reaction. Free energy profiles of the ligands diffusing through the major channel indicate that the barriers to ligand diffusion are less than 20 kJ mol−1 for each of the species. It is not clear from our study that minor channels play a role for access to the protein active site or to the protein surface.

DHR51, the Drosophila melanogaster Homologue of the Human Photoreceptor Cell-Specific Nuclear Receptor, Is a Thiolate Heme-Binding Protein

Heme has been recently described as a regulating ligand for the activity of the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta and their Drosophila homologue E75. Here, we report the cloning, expression in Escherichia coli, purification, and screening for the heme-binding ability of 11 NR ligand-binding domains of Drosophila melanogaster (DHR3, DHR4, DHR39, DHR51, DHR78, DHR83, HNF4, TLL, ERR, FTZ-F1, and E78), of unknown structure. One of these NRs, DHR51, homologous to the human photoreceptor cell-specific nuclear receptor (PNR), specifically binds heme and exhibits a UV-visible spectrum identical to that of heme-bound E75-LBD. EPR and UV-visible absorption spectroscopy indicates that, like in E75, the heme contains a hexa-coordinated low spin ferric iron. One of its axial ligands is a tightly bound cysteine, while the other one is a histidine. A dissociation constant of 0.5 microM for the heme was measured by isothermal titration calorimetry. We show that DHR51 binds NO and CO and discuss the possibility that DHR51 may be either a gas or a heme sensor.

COMPLETE AMINO ACID SEQUENCE OF PROTEUS MIRABILIS PR CATALASE. OCCURRENCE OF A METHIONINE SULFONE IN THE CLOSE PROXIMITY OF THE ACTIVE SITE

The catalase ofProteus mirabilis PR, a peroxide-resistant (PR) mutant ofProteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-typeP. mirabilis catalase.

Purification and properties of a halophilic catalase-peroxidase from Haloarcula marismortui

A heme protein, hCP, from the extreme halophile, Haloarcula marismortui, showing both peroxidatic and catalatic activity has been purified and characterized as a catalase-peroxidase. Catalatic activity is enhanced by molar concentrations of NaCl or (NH4)2SO4, while peroxidase activity decreases with increasing salt concentration. Optimal pH values are 6.0 for peroxidatic activity assayed in absence of NaCl and 7.5 for catalatic activity assayed in molar concentrations of NaCl. The two activities present saturation behaviour with increasing H2O2 concentration with apparent Km values of 0.5 and 2.5 mM for the peroxidatic and catalatic activities, respectively. A molecular mass of 81,292 +/- 9 Da was measured for the polypeptide by mass spectroscopy. One heme group (protoporphyrin IX with an iron atom in the ferric state) is associated with one molecule of hCP. Its amino-acid composition shows hCP to contain a high proportion of acidic residues. The EPR spectrum of the NO-compound of reduced (ferrous) hCP strongly suggests that the proximal ligand of the heme is the imidazole group of a histidine residue.

High Resolution Structure and Biochemical Properties of a Recombinant Proteus Mirabilis Catalase Depleted in Iron.

Heme catalases are homotetrameric enzymes with a highly conserved complex quaternary structure, and their functional role is still not well understood. Proteus mirabilis catalase (PMC), a heme enzyme belonging to the family of NADPH‐binding catalases, was efficiently overexpressed in E. coli. The recombinant catalase (rec PMC) was deficient in heme with one‐third heme and two‐thirds protoporphyrin IX as determined by mass spectrometry and chemical methods. This ratio was influenced by the expression conditions, but the enzyme‐specific activity calculated relative to the heme content remained unchanged. The crystal structure of rec PMC was solved to a resolution of 2.0 Å, the highest resolution obtained to date with PMC. The overall structure was quite similar to that of wild‐type PMC, and it is surprising that the absence of iron had no effect on the structure of the active site. Met 53 close to the essential His 54 was found less oxidized in rec PMC than in the wild‐type enzyme. An acetate anion was modeled in an anionic pocket, away from the heme group but important for the enzymatic reaction. An alternate conformation observed for Arg 99 could play a role in the formation of the H‐bond network connecting two symmetrical subunits of the tetramer. Proteins 2003;50:261–271.

SIMULATIONS OF ELECTRON TRANSFER IN THE NADPH-BOUND CATALASE FROM PROTEUS MIRABILIS PR

Catalase-bound NADPH both prevents and reverses the accumulation of compound II, an inactive form of catalase that is generated from the normal active intermediate form (compound I) when catalase is exposed to a steady flow of hydrogen peroxide. The mechanism for the regeneration reaction is unknown although NADPH could act either as a one-electron or a two-electron donor. Recently, a reaction scheme has been proposed in which the formation of compound II from compound I generates a neighboring radical species within the protein. NADPH would then donate two electrons, one to compound II for reduction of the iron and the other to the protein free radical. In this paper, we report calculations to find the dominant electron tunneling pathways between NADPH and the heme iron in the catalase from the peroxide-resistant mutant of Proteus mirabilis. Two major tunneling pathways are found which fuse together on Ser-196. It is suggested that the sequence Gly-Ser of the loop that divides the beta 5-strand is the key element for shielding a radical amino acid.

Neutron scattering from polarised proton domains

Abstract We report on time-resolved small-angle polarised neutron scattering from domains of polarised protons created by dynamic nuclear polarisation in frozen deuterated glycerol–water solutions containing a small amount of paramagnetic centres. In order to observe the rapid build-up of the polarisation of the protons around the paramagnetic ions and to separate it from the much slower polarisation change of the protons in the solvent, we have developed techniques that include stroboscopic SANS and NMR synchronised to cyclic microwave irradiation.

Polarized proton spin density images the tyrosyl radical locations in bovine liver catalase

Radical sites in catalase have been located by the method of time-resolved polarized neutron scattering from dynamically polarized protons. This method is about ten times more sensitive than magnetic neutron scattering and therefore particularly suitable for very dilute paramagnets.

Time-resolved proton polarisation (TPP) images tyrosyl radical sites in bovine liver catalase.

A differentiation between dynamic polarised protons close to tyrosyl radical sites in catalase and those of the bulk is achieved by time-resolved polarised neutron scattering. Three radical sites, all of them being close to the molecular centre and the heme, appear to be equally possible. Among these is tyr-369 the radial site of which had previously been proven by EPR.