No description available.

Mitochondrial protein biogenesis depends on the import of nucleus-encoded precursors from the cytosol. Import is highly regulated and specific for different subcompartments, with intermembrane space (IMS) import driven by an oxidative mechanism on conserved cysteine residues. Oxidative folding in the IMS is facilitated by the mitochondria import and assembly (MIA) pathway. Proteins can only be imported into the IMS in Cys-reduced unfolded forms, as oxidation prevents translocation into the IMS. How the import-competent forms are maintained in the cytoplasm is lesser characterised compared to the MIA pathway. Two recent studies suggest that the cytosolic Thioredoxin (Trx) and Glutaredoxin (Grx) reductase systems play a role in facilitating IMS protein import, with previous evidence identifying a role for yeast Trxs in small Tim protein biogenesis. In this study, the redox properties of the yeast Trx and Grx systems were investigated, as well as whether the yeast Grx system play a role in the biogenesis of two typical types of IMS precursor proteins. First, in vitro studies were carried out to determine the standard redox potentials (E°’) of the Trx and Grx enzymes. This was a quantifiable parameter of reducing activity and the results were described in Chapter 3. This study determined the E°’Trx1 value, which was shown to be a more effective reductant compared to other orthologs. Experimental limitations prevented the Grx system E°’ values being determined. Next, whether the Grx plays a role in the biogenesis of the CX3C motif-containing small Tim proteins were investigated using yeast genetic in vivo and biochemical analysis methods. The results were described in Chapter 4. There, Grxs were observed to not affect cell growth, but in using overexpressed Tim9 as an import model, significant differences were observed for the Grx system as GRX deletion significantly decreased overexpressed Tim9 levels. Study on the isolated mitochondria and cytosol with overexpressed Tim9 was unclear however. This study also observed a genetic interaction between GRX andYME1 that recovered cell functioning under respiratory conditions. Finally, whether the Grx system plays a role in the biogenesis of CX9C motif-containing proteins (Mia40, Mia40C and Cox17) was studied in Chapter 5. Whilst Mia40C (C-domain of Mia40) and Cox17 are imported into mitochondria via the MIA pathway, the full-length Mia40 is a substrate of the presequence-targeted TIM23 pathway. The results indicated that import of the full-length Mia40 was unaffected by GRX deletion. However, studies of an overexpressed Mia40C as a substrate of the MIA pathway, showed strong mitochondrial protein level decreases caused by deletion of the Grx proteins. This decrease was also accompanied by an accumulation of unimported Mia40C in the cytosol. Cox17 as an alternative MIA pathway substrate also showed decreased mitochondrial levels in the GRX deletion mutants. These results altogether suggest that the cytosolic Grx system can function in the biogenesis of CX9C motif-containing IMS proteins imported through the MIA pathway, as well as the CX3C small Tim proteins. The topic of how IMS proteins are degraded in the cell was also raised by the study of Yme1.

<p>Cytochrome <i>cd</i>₁ nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the <i>d</i>₁ haem iron of the enzyme.</p><p>Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions.</p><p>Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction.</p><p>Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty.</p><p>Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.</p>

Cytochrome cd1 nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the d1 haem iron of the enzyme. Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions. Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction. Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty. Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.