Crystal structure of the ferric homotetrameric ß4 human hemoglobin.

Spectroscopic studies carried out in the early seventies have shown that the ß-homotetramer of human hemoglobin (ß4-HbA) in the ferric state is a mixture of aquomet and bis-histidyl forms. Here we present the first crystal structure, solved at 2.10 Šresolution, of the oxidized form of ß4-HbA. The overall quaternary structure of the protein in the ferric state is virtually indistinguishable from that of the ferrous deoxygenated and carbomonoxy forms. The structure reveals that the four hemes are exclusively in an aquomet coordination, without any trace of bis-histidyl coordination. The oxidation of ß4-HbA is associated with the formation of a disulfide bridge between residues Cys112(G14) of ß1/ß4 and ß2/ß3 chains. The coordination state of ß4-HbA has been compared to that known for other organisms that exhibit bis-histidyl heme coordination in the ß4 state. This occurrence has been discussed in terms of different organism physiology.

Role of tertiary structures on the Root effect in fish hemoglobins.

Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root effect. Both tertiary and quaternary effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH6.2 and pH8.4. At low pH ligation has negligible structural effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T-R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Selective X-ray-induced NO photodissociation in haemoglobin crystals: evidence from a Raman-assisted crystallographic study.

Despite their high physiological relevance, haemoglobin crystal structures with NO bound to haem constitute less than 1% of the total ligated haemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO-ligated Hbs is most likely to be related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O(2). Here, using Raman-assisted X-ray crystallography, it is shown that under X-ray exposure (at four different radiation doses) crystals of nitrosylated haemoglobin from Trematomus bernacchii undergo a transition, mainly in the ß chains, that generates a pentacoordinate species owing to photodissociation of the Fe-NO bond. These data provide a physical explanation for the low number of nitrosylated Hb structures available in the literature.

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease.

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal ß-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.

Role of the tertiary and quaternary structure in the formation of bis-histidyl adducts in cold-adapted hemoglobins.

All tetrameric hemoglobins from Antarctic fish, including that from Trematomus bernacchii, HbTb form in the ferric state, promptly and distinctively from all the other tetrameric hemoglobins, a mixture of aquo-met at the α subunits and bis-histidyl adduct (hemichrome) at the ß subunits. The role of the tertiary and quaternary structure in the hemichrome formation is unknown. Here we report the cloning, expression, purification, spectroscopic and computational characterization of the ß-chain of HbTb (ß-HbTb). Similarly to the human ß-chains, ß-HbTb self-assembles to form the homotetramer ß(4)-HbTb; however, the latter quantitatively forms reversible ferric and ferrous bis-histidyl adducts, which are only partially present in the human tetramer (ß(4)-HbA). A molecular dynamics study of the isolated ß subunit of the two Hbs indicates that the ability to form hemichrome is an intrinsic feature of the chain; moreover, the greater propensity of ß-HbTb to form the bis-histidyl adduct is probably linked to the higher flexibility of the CD loop region. On the bases of these experimental and computational results on the isolated chain, the influence of the quaternary structure on the stability of the endogenous ferrous and ferric hexa-coordination is also discussed.

Crystallization, preliminary X-ray diffraction studies and Raman microscopy of the major haemoglobin from the sub-Antarctic fish Eleginops maclovinus in the carbomonoxy form.

The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three haemoglobins. The major haemoglobin (Hb1Em) displays the Root effect, a drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH. The carbomonoxy form of HbEm1 has been crystallized in two different crystal forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution diffraction data have been collected for both forms (1.45 and 1.49 Šresolution, respectively). The high-frequency resonance Raman spectra collected from the two crystal forms using excitation at 514 nm were almost indistinguishable. Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural characterization will shed light on the molecular mechanisms of cold adaptation and the role of the Root effect in fish haemoglobins.