The Henry Wellcome Laboratories for Biological Chemistry house one of the best-equipped laboratories in Europe for advanced magneto optical (MCD and EPR) studies of metalloenzymes. Several macromolecular protein complexes and associated model systems are studied using a wide range of current MCD and EPR techniques. We are looking to recruit a dynamic post-graduate student to work in the area of respiratory metalloproteins. Our laboratories study a wide range of respiratory proteins together with a number of groups both within UEA and throughout the world. Collaborations with groups working in the UEA Schools of Chemistry and Biological Sciences revolve around metalloenzymes involved in the nitrogen cycle, the sulphur cycles and bacterial respiration. Together with the Max-Planck Institute for Biophysics in Frankfurt, Germany (Prof. Hartmut Michel) we study a single family of enzymes, the heme copper oxidases (HCOs) which is responsible for >75% of the annual global respiratory consumption of dioxygen. Dioxygen chemistry is closely correlated with proton pumping in this class of enzymes which ultimately drives ATP synthesis. The exact nature of the proton pump driven redox events, however, is not defined since intermediates in the dioxygen chemistry reaction (termed O, R, P and F) are difficult to observe. The aim of the project is to: (A) Identify and locate local and global structural movements associated with the transitions between these O, R, P, F oxidations levels (and their substates). The motions associated with proton-pumping action will be probed using state-of-the-art pulsed EPR methods. As we have already demonstrated, the distance between two spin-labels within an oxidase can be determined using PELDOR methods. Changes in the distance between two spin-labels in response to cycling through the O-R-P-F states will be sought for strategically chosen pairs. The motion of single spin-labels relative to the paramagnetic ions of the active site will similarly be determined. (B) Determine the distribution of electrons within the active site throughout the catalytic cycle. Define the spin and oxidation state of the active site metal ions and the strength of interactions with amino-acid based radicals at each stage. It is the passage of the HCO active site through the O, R, P, F states that drives the proton-pumping mechanism yet the exact nature of these intermediates is far from clear. For example, the Pm and F states in cytochrome oxidase are both assumed to contain an active site heme A chromophore in the Fe(IV)=O state. Yet no explanation exists for the 27 nm difference in the wavelength of the characteristic visible absorption bands of these two forms. Nor is it understood why, in a bo3 oxidase, the optical differences between what are alleged to be the same two forms are negligible. Despite decades of investigation, something apparently simple such as the spin-state of the active-site heme in oxidised cytochrome aa3 is still ambiguous. These questions will be addressed using a combination of EPR spectroscopy and the novel variable-field variable-temperature MCD methods that we developed in order to successfully address the question of the exact nature of the active site in oxidised bo3.

The candidate will be given training in all aspects of biophysical spectroscopy (multi-frequency EPR and variable temperature/field MCD) which will allow them to develop and perform a successful research project within one of the aforementioned areas of respiratory metalloenzymes.

Prisner TF, Rohrer M, MacMillan F. Ann Rev Phys Chem. (2001); 52: 279.

Van Wonderen JH, Kostrz D, Dennison C, MacMillan F, Angew. Chemie (2012); under
review.

von der Hocht I, van Wonderen JH, Hilbers F, Angerer H, MacMillan F, Michel H. Proc. Natl. Acad. Sci. USA, (2011); 108: 3964-3969.

Watmough NJ, Cheesman MR, Greenwood C, Thomson AJ. Biochemical J. (1994); 300: 469-75.

MacMillan F, Kannt A, Behr J, Prisner TF, Michel H. Biochemistry (1999); 38: 9179-9184.