Redox biochemistry of Pyrococcus furiosus: Fundamental and applied aspects

Redox biochemistry of Pyrococcus furiosus: Fundamental and applied aspects

Hasan, M.N.

Hagen, W.R. (promotor)

Applied Sciences

2008-01-21

In Pyrococcus furiosus (Pfu) ferredoxin (Fd) replaces NAD+ for carrying reducing equivalent to the oxidative phosphorylation machinery, and it also takes part in the regeneration of NADPH, which is necessary for biosynthetic pathways. Therefore, it plays a central role in various metabolic pathways of the organism. PfuFd is one of the most extensively studied ferredoxins, however, some fundamental aspects remain elusive. This thesis work focuses on some of the unresolved features of the protein. Initially PfuFd was isolated as a dimer of 12-13 kDa, however, all the subsequent studies described the protein as a monomer without any experimental evidence. All (putative) natural electron-transfer partners of PfuFd are redox enzymes catalyzing two-electron reactions while ferredoxin is a one-electron carrier. It is possible that ferredoxin may interact with the redox partner enzymes in the dimeric form. Therefore, it is important to resolve the dilemma regarding the oligomeric state of the protein (chapter 2). The intra-cellular ionic strength of P. furiosus was determined to be ca. 350 mM, at which ferredoxin occurs predominantly in the dimeric form. Transition from the dimeric to monomeric form is observed at a salt concentration higher than 350 mM. We hypothesize that ferrdoxin is a dimer in vivo. PfuFd demonstrates conservation of the minimal domain containing the cluster coordinating consensus sequence. However, the cluster is coordinated by three cysteines and one aspartate residue instead of by four cysteines. In addition, two additional cysteines are present which raises the possibility of a disulfide bond formation between them. In some earlier studies the formation of a disulfide bond was demonstrated under aerobic condition and a possible contribution of the disulfide bond to the redox chemistry of the protein has been described. However, formation of a disulfide bond under the strongly reducing intracellular condition of an anaerobic organism like P. furiosus is not understandable. We demonstrate (in chapter 3) that these additional cysteines do not form a disulfide bond under intra-cellular mimicking conditions. Observing the effects of site directed mutagenesis, we attribute to the free cysteines an important structural role in the hyperthermostability of the protein. Ferredoxin, cytochrome c, rubredoxin, etc. are small electron-transfer protein and they possess well characterised metal cofactors. Electrochemistry has been successfully applied in studying the intricate properties of these electron-transfer proteins mainly in solution. However, solution voltammetry suffers from various problems like mass transport / diffusion limitation, requirement of promoters, which can be avoided by immobilizing the protein on the electrode surface. A stable and functional PfuFd immobilized gold electrode has been developed, which shows a similar electrochemical response as in the solution voltammetry. The transition between the dimeric and monomeric state on the electrode is observed in an atomic force microscopic (AFM) setup. X-ray photoelectron spectrocopy (XPS) confirms the formation of gold-thiol bonds. We find the ferredoxin electrode to be a useful tool for studying the catalytic mechanisms of the associated redox enzymes from P. furiosus. We have thus studied two redox enzymes, glyceraldehyde-3-phosphate oxidoreductase (GAPOR) and aldehyde oxidoreductase (AOR), which are involved in ferredoxin-mediated redox processes in the organism. Addition of these enzymes at room temperature results in complex formation between the electrode-bound ferredoxin and the enzyme. At 60 °C a catalytic wave appears upon addition of the substrate, glyceraldehyde-3-phosphate to the Fd-GAPOR complex. In the case of AOR at 80 °C reversible oxidation of crotonadehyde to crotonic acid and vice versa was observed. This work opens the way for the application of Fd electrodes in achieving controlled reduction of carboxylic acids on a preparative scale.

ferredoxin
electrochemistry
x-ray photoelectron spectroscopy
atomic force microscopy
ellipsometry
electrocatalysis

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978-90-9022736-8

Institutional Repository

doctoral thesis

(c) 2008 M.N. Hasan