Developing methods for study of redox proteins by combining synchrotron radiation (e.g. crystallography) and single-crystal spectroscopy

The research of the UiO Metalloprotein group at IBV (with Raman instrument at KI and electron paramagnetic resonance (EPR) instrument at FI) at UiO and staff at the Swiss-Norwegian Beam Line (SNBL) have through the later 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 biological important questions. One postdoc was 6 months in Grenoble and strengthend 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 slowly 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 a numerous of reactions e.g. used by complex IV in the respiratory chain that produce the proton gradient used for the ATP synthesis, used by cytochrome P450 to oxidize and metabolite a vast group of natural substrates and drugs, used by NO-synthase to produce the signaling molecule NO, and used by ribonucleotide reductase class I in the synthesis of deoxyribonucleotides. All of these protein systems use redox and/or metal cofactors to perform their function, some of them have tyrosyl-, tryptonyl- and thiyl-radicals. The project focus on protein-protein interaction and protein complexes. The project has one postdoctoral fellow started 1st of August 2012. This postdoctoral fellow presented a poster already in July 2012 and one other group member had a talk at the major international conference in the field called 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 medical 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 in the complex. We have published a major ribonucleotide reductase review in Coordination Chemistry Reviews with impact factor (IF) over 11 and our protein complex between R2F/NrdF and NrdI proteins from Bacillus cereus ribonucleotide was published in ACS Chem Biol. Studies were published in journals with IF over 3 of mouse, human, crucian carp, Mycobacterium tuberculosis and Bacillus cereus and of ribonucleotide reductases R2/p53R2/R2F/NrdF proteins made with electron paramagnetic resonance and Raman, in collaboration with Stanford University and CNRS in France. Furthermore a follow up EPR study was published also with IF over 3 of a copper protein with 3D-structure earlier solved at University of Tromsø in collaboration with University of Bergen, which showed presence of Cu(II) and 2 different Cu(II) sites in the secreted protein (MopE*) from methane oxidizing bacterium Methylococcus capsulatus (Bath). We published together with NMBU EPR of copper proteins in good journals. We have also improved the yield of protein production making possibly more rapid studies on protein crystals and structure. We published the first structure of Bacillus cereus NrdH (Thioredoxin BC3987) and suggested a novel reaction mechanism for some thioredoxins in a journal with IF over 3. Together with Univ. of Rochester we could show with X-ray diffraction and microspectrometry in a journal with IF 3.74 different orientation of a heme Fe(III) coordinated methionine explaining its dynamics in agreement with on going EXAFS/XAS studies with Stanford Univ. We took part in a project in which a novel mutant important for late-onset combined methylmalonic acidemia and homocystinuria was characterized. Group leader has given talks in China 2013 and Taiwan 2014 and at ZING and COST conferences. We have been active at several international meetings e.g. 16th International Conference on Biological Inorganic Chemistry in Grenoble. One researcher had two talks about combining X-Ray radiation damage, 3D structure & microspectrometry. We had 3 posters at International Union of Crystallography in Montreal 2014.

The aim is to further develop methods to be able to understand functional aspects of redox protein systems. X-ray protein crystallography (PX) determines the protein structures, but cannot give information of protonation, oxidation or spin state. For redo x proteins these properties are vital for understanding their structure and function. To be able to be in a position to answer some of the fundamental questions regarding the function of these proteins, a scheme combining several biophysical methods is es sential, especially the combination of PX and in-situ single-crystal UV-Vis and Raman spectroscopy is a very beneficial and emerging approach. During the last decade more and more focus has been put on the X-ray radiation-induced reduction of redox prote ins. Most probably are many of the structures published in the PDB not in the oxidation state that the authors originally stated. Therefore some of the challenges in this field are to have good spectroscopy setups at synchrotron beam lines for PX, so care can be taken in monitoring radiation damage, and trying to avoid it. Therefore a focus will be on understanding and develop approaches to tackle the radiation damage of redox proteins. The biological research interest will be on the functional understan ding of a few protein systems involved in oxygen activation and electron transport: the ribonucleotide reductase protein system and on the haem protein systems of NO-synthase, catalase-peroxidase, cytochrome c and myoglobin. Key challenges involving these protein systems are to both understand the systems on an electronic level for elucidating the detailed reaction mechanism, as well as the interaction between the different proteins.