Biomedical subjects

Estelle M Maes

Publications and source records attributed to Estelle M Maes.

5 recordsLinked to original sources

PubMed2005

Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes.

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.

Carbon Monoxide↗

PubMed2005

Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein.

Certain bloodsucking insects deliver nitric oxide (NO) while feeding, to induce vasodilation and inhibit blood coagulation. We have expressed, characterized, and determined the crystal structure of the Cimex lectularius (bedbug) nitrophorin, the protein responsible for NO storage and delivery, to understand how the insect successfully handles this reactive molecule. Surprisingly, NO binds not only to the ferric nitrophorin heme, but it can also be stored as an S-nitroso (SNO) conjugate of the proximal heme cysteine (Cys-60) when present at higher concentrations. EPR- and UV-visible spectroscopies, and a crystallographic structure determination to 1.75-A resolution, reveal SNO formation to proceed with reduction of the heme iron, yielding an Fe-NO complex. Stopped-flow kinetic measurements indicate that an ordered reaction mechanism takes place: initial NO binding occurs at the ferric heme and is followed by heme reduction, Cys-60 release from the heme iron, and SNO formation. Release of NO occurs through a reversal of these steps. These data provide, to our knowledge, the first view of reversible metal-assisted SNO formation in a protein and suggest a mechanism for its role in NO release from ferrous heme. This mechanism and Cimex nitrophorin structure are completely unlike those of the nitrophorins from Rhodnius prolixus, where NO protection is provided by a large conformational change that buries the heme nitrosyl complex, highlighting the remarkable evolution of proteins that assist insects in bloodfeeding.

Animals↗

PubMed2004

Structural dynamics controls nitric oxide affinity in nitrophorin 4.

Nitrophorin 4 (NP4) is one of seven nitric oxide (NO) transporting proteins in the blood-sucking insect Rhodnius prolixus. In its physiological function, NO binds to a ferric iron centered in a highly ruffled heme plane. Carbon monoxide (CO) also binds after reduction of the heme iron. Here we have used Fourier transform infrared spectroscopy at cryogenic temperatures to study CO and NO binding and migration in NP4, complemented by x-ray cryo-crystallography on xenon-containing NP4 crystals to identify cavities that may serve as ligand docking sites. Multiple infrared stretching bands of the heme-bound ligands indicate different active site conformations with varying degrees of hydrophobicity. Narrow infrared stretching bands are observed for photodissociated CO and NO; temperature-derivative spectroscopy shows that these bands are associated with ligand docking sites close to the extremely reactive heme iron. No rebinding from distinct secondary sites was detected, although two xenon binding cavities were observed in the x-ray structure. Photolysis studies at approximately 200 K show efficient NO photoproduct formation in the more hydrophilic, open NP4 conformation. This result suggests that ligand escape is facilitated in this conformation, and blockage of the active site by water hinders immediate reassociation of NO to the ferric iron. In the closed, low-pH conformation, ligand escape from the active site of NP4 is prevented by an extremely reactive heme iron and the absence of secondary ligand docking sites.

Animals↗

PubMed2004

Role of binding site loops in controlling nitric oxide release: structure and kinetics of mutant forms of nitrophorin 4.

Nitrophorins are ferric heme proteins that transport nitric oxide (NO) from blood-sucking insects to victims. NO binding is tighter at lower pH values, as found in the insect salivary gland, and weaker at the pH of the victim's tissue, facilitating NO release and subsequent vasodilation. Previous structural analyses of nitrophorin 4 (NP4) from Rhodnius prolixus revealed a substantial NO-induced conformational change involving the A-B and G-H loops, which rearrange to desolvate the distal pocket and pack nonpolar residues against the heme-ligated NO. Previous kinetic analyses revealed a slow, biphasic, and pH-dependent NO release, which was proposed to be associated with loop movements. In this study, we created NP4 mutants D30A and D30N (A-B loop), D129A/L130A (G-H loop), and T121V (distal pocket). Eight crystal structures were determined, including complexes with NO, NH(3), and imidazole, to resolutions as high as 1.0 A. The NO-induced conformational change is largely abolished in the loop mutants, but retained in T121V. Kinetic analyses using stopped-flow spectroscopy revealed the pH dependence for NO release is eliminated for D129A/L130A, considerably reduced for D30A and D30N, but retained for T121V. NO association rates were increased 2-5-fold for T121V, but were unchanged in the loop mutants. Taken together, our findings demonstrate that the pH dependency for NO release is linked to loop dynamics and that solvent reorganization is apparently rate-limiting for formation of the initial iron-nitrosyl bond. Interestingly, the multiphasic kinetic behavior of rNPs was not affected by mutations, and its cause remains unclear.

Binding Sites↗

PubMed2002

Removal of a cysteine ligand from rubredoxin: assembly of Fe(2)S(2) and Fe(S-Cys)(3)(OH) centres.

The electron transfer protein rubredoxin from Clostridium pasteurianum contains an Fe(S-Cys)(4) active site. Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichia coli. The C42A protein expresses with a Fe(III)(2)S(2) cluster in place. In contrast, the other proteins are isolated in colourless forms, although a Fe(III)(2)S(2) cluster may be assembled in the C42G protein via incubation with Fe(III)and sulfide. The four mutant proteins were isolated as stable mononuclear Hg(II)forms which were converted to unstable mononuclear Fe(III)preparations that contain both holo and apo protein. The Fe(III)systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies. The dominant Fe(III) form in the C9A preparation is a Fe(S-Cys)(3)(OH) centre, similar to that observed previously in the C6S mutant protein. Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells. In addition to Fe(S-Cys)(3)(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate Fe(III)form in which a ligand appears to be supplied by the protein chain.

Amino Acid Substitution↗