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The Pfizer Award in Enzyme Chemistry is an award given by the Division of Biological Chemistry of the American Chemical Society C. Since 1945 it has been awarded to researchers under the age of 40 to "recognise outstanding work in enzyme chemistry where the presence of enzyme action is unequivocally demonstrated." D The 2008 Pfizer Award was presented to Carsten Krebs for two main areas of research that were the subject of several journal articles published between 2003 and 2007. The first of these projects focuses on mononuclear non-heme-iron enzymes that activate O2, namely the Fe(II)/ ¡-ketogluturate-dependent dioxygenases. The second project that led to the award was the discovery of the Mn/Fe cofactor in Chlamidia trachomatis Ribonucleotide Reductase. This essay will discuss these two projects in turn.
Carsten Krebs obtained his bachelor's degree and diploma degree from Ruhr-Universitat Bochum, Germany in 1991 and 1994 respectively. He achieved his doctoral degree in inorganic chemistry at the Max Planck Institute for Radiation Chemistry in 1997 and in 2001 completed a postdoctoral fellowship at Emory University, U.S.A. He is currently an Assistant Professor in the Department of Biochemistry and Molecular Biology and the Department of Chemistry at The Pennsylvania State University, U.S.A. His other awards include the Young Investigator Award from the Arnold and Mabel Beckman Foundation in 2005 and a Camille Dreyfus Teacher-Scholar Award from the Camille and Henry Dreyfus Foundation in 2006 A+E.
Carsten Krebs' expertise in spectroscopy and inorganic chemistry is complemented by J. Martin Bollinger Jr.'s expertise in biochemistry and kinetics. Together they have a joint group which concentrates on the mechanisms of metalloenzymes.
Fe(II)/ ¡-ketogluturate (¡KG) -dependent dioxygenases
Fe(II)/ ¡-ketogluturate (¡KG) -dependent dioxygenases are found in animals, plants, protists, fungi, bacteria and viruses.2 They are a large family of enzymes that generally use the oxidative decomposition of ¡KG to hydroxylate unactivated carbon atoms on a large range of substrates.1 Some members of this group of enzymes have also been shown to catalyse many other reactions such as halogenation, epoxidation and decarboxylation.2 This variety of performed reactions mean that they play a variety of important roles in biological systems. The enzymes are key in the synthesis of penicillin3, repair of alkylated DNA strands and are involved in fatty acid metabolism.6 The also play an inportant role in the hyposic response pathway5. Fe(II)/ ¡KG -dependent dioxygenases all contain a mononucluler non-heme iron cofactor. This iron centre is most commonly coordinated by two histidines and an aspartic acid/glutamic acid residue, which form the "facial triad" that occupies one face of an octohedron. The three remaining sites are then used during the reaction.B The majority of study that earned Carsten Krebs the 2008 Pfizer award in enzyme chemistry was performed on the enzyme taurine/¡-ketogluturate dioxygenase (TauD) from Escherichia Coli. The crystal structure of this enzyme was published in 2002, and is shown in figure ___.7
Like other members of this family, TauD has a core motif of eight b sheets in a "jellyroll" manner. TauD catalyses the hydroxylation of the taurine (and other sulphonates) to sulphite and aminoaldehyde. By 2003, a consensus mechanism for this process had been reached (scheme___) until Krebs et al made the first direct characterisation of a high-valent iron intermediate in this reaction.8 This result was obtained using stopped-flow absorption and freeze-quench Mossbauer and EPR experiments. The stopped-flow absorption spectra give experimental evidence for two intermediates in this reaction. The first has a strong absorption feature at 318nm. With its decay, a second intermediate is formed which has an absorption feature at 520nm, which coincidentally is the same wavelength as seen for the starting quaternary complex. The secondary intermediate and the initial complex can be distinguished by looking at difference spectra. As the intermediate is weakly absorbing, a negative feature is observed at 520nm, which returns to a featureless spectrum upon decay back to the quaternary complex. Mossbauer spectroscopic investigations found an intermediate species with a quadrapole doublet of ¤ = 0.31 mm s-1 for the isomer shift. It also found that the intermediate must be paramagnetic with an integer-spin ground state of S (greater or equal to 2). These observations allowed unambiguous assignment of a high spin Fe(IV) intermediate.
Although the isomer shift is quite large for an Fe(IV) complex, the ligands around the iron centre play a crucial role in absorbing the high valence state and confer some Fe(III) character on the intermediate.
Kinetic competency of this intermediate was tested by cryoreduction of the intermediate to a high spin Fe(III) complex. In this procedure (which can also be used to perform a one electron reduction on the Q cluster of methane monooxygenase) electrons are released from glycerol by rays that can reduce the Fe centre whilst keeping the geometry intact due to the liquid nitrogen temperatures used. The freeze quench Mossbauer spectrum 20ms after reduction shows a significant decrease in the signals of the first intermediate, but no change in the signal that corresponds to the starting complex. Although the new peaks for the proposed Fe(III) reduced species were not well defined, the high spin Fe(III) state was confirmed by EPR spectroscopy. These observations lead them to propose a new kinetic mechanism which is shown in scheme_____.
Following on from this work later that year, the group showed that the first intermediate is most likely involved in the C1 taurine hydrogen abstraction step by using isotopic labelling.9 Dueteration at C1 of taurine made no difference to the absorption features in the stopped flow experiments, however the first intermediate persisted significantly longer. In fact, the kinetic isotope effect caused a roughly 37 fold decrease in reaction time. As the C1 hydrogen position of taurine is prochiral this value is thought to have contributions from both primary and secondary kinetic isotope effects. Accumulation of the secondary intermediate increased to 60%. This dramatic increase of lifetime opened up new avenues for spectroscopic analysis and in 2004, the team published compelling evidence that the first intermediate has a Fe=O unit.10 This evidence came in the form of EXAFS and XANES experiments. The XANES experiment showed that the intermediate must be more oxidised than the Fe(II) centre of the starting quaternary complex, as its absorption edge is significantly higher in energy. Although an oxidation state of 4+ could not be ambiguously assigned (due to the aforementioned Fe(III) character), the general shape and the energy (7123 eV) of the curve are similar to literature examples of Fe(IV) protein intermediates). The most solid evidence comes from the fitting of the EXAFS oscillations, which require a Fe-O bond of 1.62 angstroms which is characteristic of a ferryl group. This finding is also supported by the resonance Raman spectroscopy findings by Hausinger et al a few months before.11
The final project undertaken by Krebs et al on this family of enzymes that lead to the 2008 Pfizer award was focused on P4H, an ¡KG -dependent prolyl-4-hydroxylase, from Chlorella virus 1.12 P4H catalyses hydroxylation at C4 of proline. These 4-hydroxyproline residues are essential at stabilising the collagen triple helix25, and regulate the activity of hypoxia inducible factor (HIF).13 In 2006, the group used their experience with the TauD enzyme and applied similar analysis to P4H, using (Pro-Ala-Pro-Lys)3 as the substrate peptide. Stopped-flow kinetics showed very similar results as with TauD with the first intermediate showing a strong absorption at 320nm. The starting quaternary complex had the same absorption at 520nm that the second intermediate has, but again the second intermediate is weakly absorbing. When both prolines are deuterated (d7-Pro-Ala-d7Pro-Lys)3 a large KIE of around 60 for the first intermediate was found. Although this is higher than that found for TauD, the authors consider the P4H value an estimate, as the decay of the first intermediate in the protic reaction is too rapid. They do however feel that the result is accurate enough to show that the first intermediate is involved in the hydrogen abstraction step, as for TauD. The Mossbauer spectra for P4H and TauD are remarkably similar with a quadrapole doublet of ¤ = 0.30 mm s-1 for the first intermediate of P4H (¤ = 0.31mm s-1 for TauD) (Figure______). Again the Mossbauer results give a necessary state of S (greater or equal to) 2 for the first intermediate, indicative of a Fe(IV) centre. These results show that even distantly related enzymes in the same family have the same conserved mechanism.
Chlamydia trachomatis ribonucleotide reductase
The second general project for which Dr. Krebs was awarded the 2008 Pfizer award for concerned for work done on the enzyme Chlamydia trachomatis ribonucleotide reductase.14 Ribonucleotide reductases (RNRs) catalyse the substitution of the 2'OH on ribonucleotides to a hydrogen atom to give deoxyribonucleotides.15 RNRs are split into 3 classes, based on their mechanism of action. Class I RNRs such as those found in Homo Sapiens, have a binuclear iron centre in the R2 cofactor subunit. This centre oxidises a neighbouring tyrosine residue, which, by long distance electron transfer, generates a cystine radical located within the R1 subunit which can then reduce the substrate ribonucleotide.16 A major exception to this general rule is that of Chlamydia trachomatis ribonucleotide reductase (Figure ____) whose R2 subunit has a phenylalanine residue in place of the aforementioned tyrosine residue.17
When the crystal structure was solved by Nordlund et al, the binuclear centre of the cofactor was assigned as containing two iron atoms, however in 2005 Krebs et al discovered a lack of correlation between catalytic activity and iron contents of different preparations of R2.14 Iron was removed using EDTA as a chelating agent and upon addition of two equivalents of Fe(II) catalytic activity did not resume at a significant rate. However, addition of a 1:1 Mn(II):Fe(II) mixture gave catalytic activity of greater than 85% of the maximum rate. They also showed that the enzyme could not turn over under anaerobic conditions. EPR spectroscopy gave no obvious signal for the active form but a Mossbauer quadrupole doublet with an isomer shift of ¤ = 0.52 mm s-1 establishes that the iron centre becomes Fe(III) upon addition of oxygen. Reduction of the complex with dithionite makes little difference to the Mossbauer parameters, demonstrating that the iron centre stays in the 3+ oxidation state. Hyperfine coupling observed in the Mossbauer spectrum of the reduced species show hyperfine coupling between the I = 5/2 55Mn nucleus and the I = 1/2 57Fe nucleus. This means that the reduced Mn site must have an even number of electrons to couple the odd electron on Fe(III) so Mn(III) must be the reduced form. These findings lead the team to assign a Mn(IV)/Fe(III) cofactor to Chlamydia trachomatis ribonucleotide reductase, which was unknown as a cofactor for radical initiation until this point. Further work done the same year using Mossbauer spectroscopy showed that the cofactor has a triplet ground state which results from antiferromagnetic coupling of its Mn(IV) (SMn = 3/2) and high-spin Fe(III) (SFe = 5/2) centres.18 Finally in 2007 the team demonstrated that during the formation of the Mn(IV)/Fe(III) active form by O2 from Mn(II)/Fe(II) goes via a Mn(IV)/Fe(IV) intermediate.19 This relatively long lived intermediate gives an g = 2 EPR signal that shows a hyperfine coupling between 55Mn and 57Fe. The decay of the intermediate to the enzymatic active form of Mn(IV)/Fe(III) is quickened by the addition of ascorbate (a one electron reductant), which implies that the intermediate is more oxidised than the active complex. These results are also supported by Mossbauer spectroscopic studies.
Carsten Krebs was awarded the 2008 Pfizer Award in Enzyme Chemistry for his outstanding contributions towards the understanding of the TauD mechanism and for the discovery of a Mn(IV)/Fe(III) based cofactor within Chlamydia trachomatis ribonucleotide reductase. Work done after the prize was awarded showed that the enzyme could be rapidly activated upon the addition of hydrogen peroxide.20 They also probed the structure of this binuclear centre using EXAFS experiments.21
Current work within the Krebs/ Bollinger Jr. group concentrates on cyanobacterial aldehyde decarbonylases which they believe to have significant potential in the production of alternative energy. They discovered that formate is produced stoiciometrically during the transformation of fatty acids into alkenes by these enzymes22 and are currently probing the mechanism of this catalysis. 23,24