Hans-Petter Hersleth

Structure of the haptoglobin–haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin–haemoglobin complex, which represents a virtually irreversible non-covalent protein–protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin–haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin–haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin–haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin–haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin–haemoglobin for CD163 (ref. 4).

Review Studies of Ferric Heme Proteins with Highly Anisotropic/Highly Axial Low Spin (S = 1/2) Electron Paramagnetic Resonance Signals with bis-Histidine and Histidine-Methionine Axial Iron Coordination

Six‐coordinated heme groups are involved in a large variety of electron transfer reactions because of their ability to exist in both the ferrous (Fe2+) and ferric (Fe3+) state without any large differences in structure. Our studies on hemes coordinated by two histidines (bis‐His) and hemes coordinated by histidine and methionine (His‐Met) will be reviewed. In both of these coordination environments, the heme core can exhibit ferric low spin (electron paramagnetic resonance EPR) signals with large gmax values (also called Type I, highly anisotropic low spin, or highly axial low spin, HALS species) as well as rhombic EPR (Type II) signals. In bis‐His coordinated hemes rhombic and HALS envelopes are related to the orientation of the His groups with respect to each other such that (i) parallel His planes results in a rhombic signal and (ii) perpendicular His planes results in a HALS signal. Correlation between the structure of the heme and its ligands for heme with His‐Met axial ligation and ligand‐field parameters, as derived from a large series of cytochrome c variants, show, however, that for such a combination of axial ligands there is no clear‐cut difference between the large gmax and the “small g‐anisotropy” cases as a result of the relative Met‐His arrangements. Nonetheless, a new linear correlation links the average shift 〈δ〉 of the heme methyl groups with the gmax values.

Crystallographic and Spectroscopic Studies of Peroxide-derived Myoglobin Compound II and Occurrence of Protonated FeIV–O

High resolution crystal structures of myoglobin in the pH range 5.2–8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe–O bond length (1.86–1.90 Å) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe–O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an FeIV–O species with a single bond. The presence of FeIV is indicated by a small isomer shift of δ = 0.07 mm/s from Mössbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated.

The Crystal Structure of Peroxymyoglobin Generated Through Cryoradiolytic Reduction of Myoglobin Compound III During Data Collection.

Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 A (1 A=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 A and O-O bond of 1.3 A in accordance with a Fe(II)-O-O- (or Fe(III)-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.

How different oxidation states of crystalline myoglobin are influenced by X-rays

X-ray induced radiation damage of protein crystals is well known to occur even at cryogenic temperatures. Redox active sites like metal sites seem especially vulnerable for these radiation-induced reductions. It is essential to know correctly the oxidation state of metal sites in protein crystal structures to be able to interpret the structure-function relation. Through previous structural studies, we have tried to characterise and understand the reactions between myoglobin and peroxides. These reaction intermediates are relevant because myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress, and because these intermediates are similar among the haem peroxidases and oxygenases. We have in our previous studies shown that these different myoglobin states are influenced by the X-rays used. In this study, we have in detail investigated the impact that X-rays have on these different oxidation states of myoglobin. An underlying goal has been to find a way to be able to determine mostly unreduced states. We have by using single-crystal light absorption spectroscopy found that the different oxidation states of myoglobin are to a different extent influenced by the X-rays (e.g. ferric Fe(III) myoglobin is faster reduced than ferryl Fe(IV)═O myoglobin). We observe that the higher oxidation states are not reduced to normal ferrous Fe(II) or ferric Fe(III) states, but end up in some intermediate and possibly artificial states. For ferric myoglobin, it seems that annealing of the radiation-induced/reduced state can reversibly more or less give the starting point (ferric myoglobin). Both scavengers and different dose-rates might influence to which extent the different states are affected by the X-rays. Our study shows that it is essential to do a time/dose monitoring of the influence X-rays have on each specific redox-state with spectroscopic techniques like single-crystal light absorption spectroscopy. This will determine to which extent you can collect X-ray diffraction data on your crystal before it becomes too heavily influenced/reduced by X-rays. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

The Influence of X-Rays on the Structural Studies of Peroxide-Derived Myoglobin Intermediates

In recent years, the awareness of potential radiation damage of metal centers in protein crystals during crystallographic data collection has received increasing attention. The radiation damage can lead to radiation‐induced changes and reduction of the metal sites. One of the research fields where these concerns have been comprehensively addressed is the study of the reaction intermediates of the heme peroxidase and oxygenase reaction cycles. For both the resting states and the high‐valent intermediates, the X‐rays used in the structure determination have given undesired side effects through radiation‐induced changes to the trapped intermediates. However, X‐rays have been used to generate and trap the peroxy/hydroperoxy state in crystals. In this review, the structural work and the influence of X‐rays on these intermediates in myoglobin are summarized and viewed in light of analogous studies on similar intermediates in peroxidases and oxygenases.

Structural Characterization of Nitrosomonas Europaea Cytochrome C-552 Variants with Marked Differences in Electronic Structure.

Nitrosomonas europaea cytochrome c‐552 (Ne c‐552) variants with the same His/Met axial ligand set but with different EPR spectra have been characterized structurally, to aid understanding of how molecular structure determines heme electronic structure. Visible light absorption, Raman, and resonance Raman spectroscopy of the protein crystals was performed along with structure determination. The structures solved are those of Ne c‐552, which displays a “HALS” (or highly anisotropic low‐spin) EPR spectrum, and of the deletion mutant Ne N64Δ, which has a rhombic EPR spectrum. Two X‐ray crystal structures of wild‐type Ne c‐552 are reported; one is of the protein isolated from N. europaea cells (Ne c‐552n, 2.35 Å resolution), and the other is of recombinant protein expressed in Escherichia coli (Ne c‐552r, 1.63 Å resolution). Ne N64Δ crystallized in two different space groups, and two structures are reported [monoclinic (2.1 Å resolution) and hexagonal (2.3 Å resolution)]. Comparison of the structures of the wild‐type and mutant proteins reveals that heme ruffling is increased in the mutant; increased ruffling is predicted to yield a more rhombic EPR spectrum. The 2.35 Å Ne c‐552n structure shows 18 molecules in the asymmetric unit; analysis of the structure is consistent with population of more than one axial Met configuration, as seen previously by NMR. Finally, the mutation was shown to yield a more hydrophobic heme pocket and to expel water molecules from near the axial Met. These structures reveal that heme pocket residue 64 plays multiple roles in regulating the axial ligand orientation and the interaction of water with the heme. These results support the hypothesis that more ruffled hemes lead to more rhombic EPR signals in cytochromes c with His/Met axial ligation.

Crystal Structure of Bacillus Cereus Class Ib Ribonucleotide Reductase Di-Iron Nrdf in Complex with Nrdi.

Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (β2) subunit to initiate ribonucleotide reduction in the NrdE (α2) subunit. Contrary to the diferric tyrosyl radical (Fe(III)2-Y•) cofactor, which can self-assemble from Fe(II)2-NrdF and O2, generation of the Mn(III)2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-Mn(II)2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein-protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus Mn(II)2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the dimetal site and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF dimetal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested α2β2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved.

Selective solvent inclusion as a tool for mapping molecular properties in crystal structures: a diethylstilbestrol example

Useful information about hydrogen bonding, the preferred modes of hydrophobic interaction and conformational preferences of a specific molecule can be obtained from cocrystallization of the solute with a selected series of solvent molecules. This method is used in a study of nine different crystal structures of diethylstilbestrol (DES) solvates. It is shown that solvent inclusion results not only in stronger hydrogen bonds, but usually also in a larger number of favorable C-H.pi interactions between DES molecules. Furthermore, solvent molecules such as DMSO, DMF, acetonitrile and acetone demonstrate important hydrogen-bond donating properties in addition to their more familiar role as hydrogen-bond acceptors. Molecular conformations in the crystal structures compare favorably with results from molecular mechanics calculations.

Installation of an IR/Raman measuring station at the ESRF for simultaneous detection of vibrational and nuclear resonant scattering spectra

Research on micro-structured iron-containing samples can be efficiently carried out by means of nuclear resonant scattering techniques at synchrotron facilities, because the high brilliance and low divergence of the beam facilitates the characterization of micrometer-sized samples containing Mossbauer active isotopes. Additional information can be obtained if the nuclear scattering techniques are combined with classical vibrational methods such as Raman and IR microspectroscopy. The sample environment presented within this paper provides the possibility to measure Raman spectra and to obtain optical (visible) on-line information about possible changes/damages of the sample during synchrotron beam exposition.