3D protein structure, active site structure, and function of metallo- and radical proteins

The research performed by the Metalloprotein group at IBV, UiO (including a Raman instrument at KI and an electron paramagnetic resonance (EPR) instrument at FI), and staff at the Swiss-Norwegian Beam Line (SNBL), have during recent years taken a lead in developing and using in-situ single-crystal spectroscopic methods in combination with protein crystallography at the synchrotrons. The current project focuses on further developing these methods, and the use of them to solve important biological questions. One PhD student has been at Stanford University (Dept. of Chemistry) during end of 2013 and most of 2014 for advanced magnetic studies of metalloproteins. One postdoc stayed six months in Grenoble /ESRF and improved the instrumentation setup at SNBL, further developing the use of the combined single-crystal spectroscopy and protein crystallography methods. X-ray protein crystallography determines the protein structures, but cannot give information of protonation state, oxidation state or spin state. For metallo- and redox proteins, these properties are essential for understanding their structure and function. During the last decade more focus has been put on the X-ray radiation induced reduction of these enzymes. We focus on enzymes involved in oxygen activation and electron transport, which are of the most important processes in life. To understand processes like respiration and photosynthesis, it is vital to understand the electron transport within and among these systems. Oxygen is activated and utilized in numerous reactions, e.g. by complex IV in the respiratory chain producing the proton gradient used for ATP synthesis. It is used by cytochrome P450 to oxidize and metabolize a vast group of natural substrates and drugs. Electrons and oxygen are also used by ribonucleotide reductase (RNR) class I in the synthesis of deoxyribonucleotides. All of these systems use redox and/or metal cofactors to perform their function, some of them including tyrosyl-, tryptonyl- and thiyl-radicals. The project focuses on protein-protein interactions and protein complexes. Dr. Åsmund K. Røhr presented a poster already in July 2012, and another group member gave a talk at the major international conference Simultaneous Combination of Spectroscopies with X-ray Absorption, Scattering and Diffraction Techniques in Zürich (CH). The project has had several publications with high impact. The group was part of a publication in the outstanding journal Nature, where the crystal structure of the medically relevant haptoglobin-haemoglobin/hemoglobin complex was published. We contributed with our expertise in UV-vis and Raman single protein crystal spectroscopies, showing that the complex was in its oxygenated state. We have published a major RNR review in Coordination Chemistry Reviews with impact factor (IF) above 12, and our protein complex of R2F/NrdF with NrdI from Bacillus cereus RNR was published in ACS Chem. Biol. (IF above 5). Several of our studies have been published in journals with IF above 3. These include studies of mouse, human, crucian carp, Mycobacterium tuberculosis and Bacillus cereus RNR, as well as electron paramagnetic resonance and Raman spectroscopic studies of RNR R2/p53R2/R2F/NrdF proteins, in collaboration with Stanford University and CNRS in France. Furthermore, a follow-up EPR study was published (IF above 3) of a copper protein with 3D-structure earlier solved at University of Tromsø, in collaboration with University of Bergen, showing the presence two different Cu(II) sites in the secreted protein (MopE*) from the methane oxidizing bacterium Methylococcus capsulatus (Bath). Together with NMBU, we have published EPR data on copper proteins in a good journal. We have also improved the yield of protein production, likely enabling more rapid crystallographic studies. We published the first structure of Bacillus cereus NrdH (thioredoxin BC3987) and suggested a novel reaction mechanism for a particular type of thioredoxins (IF above 3). Together with Univ. of Rochester we could show with X-ray diffraction and microspectrometry, in a journal with IF 3.74, different orientations of a heme Fe(III) coordinated methionine explaining its dynamics in agreement with ongoing EXAFS/XAS studies with Stanford Univ. In Chemical Commutations (IF 6.8), in collaboration with CUNY in New York, we published important findings showing that in KatG heme is open to one substrate. The group leader has given talks in China (2013), Taiwan (2014) and India (2017) at the ZING and COST conferences. We have attended several international meetings. Two of our master students; Susanne Monka and Bernt Wu, finished their theses in 2015. PhD student Marie Lofstad defended her thesis 3 Feb, 2017 with one USA and one Spain as judges. Marie has results from Stanford and published in Biochemistry as first author. Together with Univ. of Vienna and others, we have submitted cancer related studies with Dr. Marta Hammerstad.

The aim of the project is to investigate structural features of oxygen or electron reactions in proteins containing functionally important iron/manganese/cobalt, and/or radical centers. We will study mono and other (per)oxygenases that can perform comple x reactions, and ribonucleotide reductases (RNR) with related proteins (e.g. NrdI, NrdH), well as cytochromes with unusual EPR properties that could indicate original electron and proton coupled radical (in RNR) transfer reactions. Monooxygenases, perox idases, oxygenases, RNR and heme proteins can have large medical importance, e.g. tetrahydrobiopterin dependent monooxygenases/ heme proteins nitric oxide synthase, RNR and myoglobin/hemoglobin mutations are all linked to illnesses. We plan to study: 1) t he ferryl versus hydroxy-ferryl species and radical formation in myoglobin 2) nitric oxide synthase and other oxygenases 3) peroxide/superoxide complexes in heme protein and from non-heme iron containing proteins; 4) mono-nuclear iron dependent proteins will be compared with di-nuclear proteins and/or with Mn, Co proteins; and 5) bacterial cytochromes with unusual electronic properties. 6) the Met-Tyr-Trp cofactor realation to reactive oxygen/peroxide derived species will be determined. Nitric oxide sy nthase, peroxidase-catalase KatG mutants are cloned and expressed in E. coli and carp RNR mutants will be obtained, while some proteins are isolated in native form. The long-term goal is the understanding of monooxygenase and radical chemistry important t o make rational drug or modulator design. We are developing a combination of techniques for proteins in solution and in crystal form. We have progress in EPR and resonance Raman studies of protein crystals. We continue to develop (HF EPR, single crystal v ibrational Mössbauer, Raman, EXAFS/XAS/DFT analysis) of radicals and metal sites also after irradiation, and under hypoxia/anoxia. The goals of this project strongly relate to sustainable development of our planet.