K. Kristoffer Andersson

The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein

A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has approximately 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMO(m)), using the non-ionic detergent dodecyl-beta-D-maltoside, to yield solubilized enzyme (pMMO(s)). Further purification gave rise to an active complex (pMMO(c)) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24 kDa subunits) and the pMMOR (63 and 8 kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with (63)Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe(3+) ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.

Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate-active enzyme classification system, are copper-dependent enzymes that oxidize sp(3)-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality.

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.

The role of tetrahydrobiopterin in the activation of oxygen by nitric-oxide synthase.

We have studied the reaction of reduced nitric-oxide synthase (NOS) with molecular oxygen at -30 degrees C. In the first reaction cycle (from L-Arg to hydroxy-L-Arg), an oxygen adduct complex formed rapidly. Experiments in the absence of the reductase domain demonstrated that this complex was then further reduced by one electron stemming from the cofactor tetrahydrobiopterin (BH4). Spectral evidence suggested an iron(IV) porphyrin pi-cation radical as an intermediate. The nature of the oxidized BH4 was identified by EPR as a BH3* radical. Within the second cycle (from hydroxy-L-Arg to citrulline and NO), an iron(III)-NO complex could be identified clearly by its spectral characteristics. The strict requirement of BH4 for its formation suggests that BH4 plays a redox role, although transient, also in the second reaction cycle.

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 use of high field/frequency EPR in studies of radical and metal sites in proteins and small inorganic models.

Low temperature electron paramagnetic resonance (EPR) spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T have been used to study radicals and metal sites in proteins and small inorganic model complexes. We have studied radicals, Fe, Cu and Mn containing proteins. For S = 1/2 systems, the high frequency method can resolve the g-value anisotropy. It was used in mouse ribonucleotide reductase (RNR) to show the presence of a hydrogen bond to the tyrosyl radical oxygen. At 285 GHz the type 2 Cu(II) signal in the complex enzyme laccase is clearly resolved from the Hg(II) containing laccase peroxide adduct. For simple metal sites, the systems over S = 1/2 can be described by the spin Hamiltonian: H(S) = BgS + D[Sz2 - S(S + 1)/3 + E/D (Sx2 - Sy2)]. From the high frequency EPR the D-value can be determined directly by, (I) shifts of g(eff) for half-integer spin systems with large D-values as observed at 345 GHz on an Fe(II)-NO-EDTA complex, which is best described as S = 3/2 system with D = 11.5 cm(-1), E = 0.1 cm(-1) and gx = gy = gz = 2.0; (II) measuring the outermost signal, for systems with small D values, distant of (2S - 1) x absolute value(D) from the center of the spectrum as observed in S= 5/2 Fe(III)-EDTA. In Mn(II) substituted mouse RNR R2 protein the weakly interacting Mn(II) at X-band could be observed as decoupled Mn(II) at 285 GHz.

NrdH-Redoxin Protein Mediates High Enzyme Activity in Manganese-reconstituted Ribonucleotide Reductase from Bacillus anthracis

Background: Class Ib ribonucleotide reductase of the severe pathogen Bacillus anthracis can be loaded with manganese or iron. Results: The manganese form was 10-fold more active than the iron form in the presence of the physiological protein NrdH-redoxin. Conclusion: The manganese form is important in the life cycle of B. anthracis. Significance: The physiologically relevant form of ribonucleotide reductase controls B. anthracis proliferation and survival. Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn2III site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe2III site (Fe-NrdF) formed spontaneously from Fe2+ and O2. In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

Deciphering complex protein interaction kinetics using Interaction Map

Cellular receptor systems are expected to present complex ligand interaction patterns that cannot be evaluated assuming a simple one ligand:one receptor interaction model. We have previously evaluated heterogeneous interactions using an alternative method to regression analysis, called Interaction Map (IM). IM decomposes a time-resolved binding curve into its separate components. By replacing the reductionistic, scalar kinetic association rate constant k(a) and dissociation rate constant k(d) with a two-dimensional distribution of k(a) and k(d), it is possible to display heterogeneous data as a map where each peak corresponds to one of the components that contribute to the cumulative binding curve. Here we challenge the Interaction Map approach by artificially generating heterogeneous data from two known interactions, on either LigandTracer or Surface Plasmon Resonance devices. We prove the ability of IM to accurately decompose these man-made heterogeneous binding curves composed of two different interactions. We conclude that the Interaction Map approach is well suited for the analysis of complex binding data and forecast that it has a potential to resolve previously uninterpretable data, in particular those generated in cell-based assays.