The X-ray crystal structures of cytochrome cL and methanol dehydrogenase from Methylobacterium extorquens

The soluble proteins involved in the oxidation of methanol in the methylotrophic bacteria Methylobacterium extorquens provide an ideal system in which to study electron transfer. Methanol is oxidized to formaldehyde by methanol dehydrogenase (MDH), reducing the pyrroloquinoline quinone (PQQ) prosthetic group to quinol. Electrons are then passed, one at a time, to the c-type cytochrome, cytochrome cL, creating the semiquinone PQQ free radical after the first transfer. Cytochrome cL is reoxidised by the typical class I c-type cytochrome, cytochrome cH, which in turn passes electrons to the terminal electron acceptor, the membrane bound cytochrome oxidase, cytochrome aa3. The crystal structures of MDH and cytochrome cH have been solved prior to this project. The principal aim of this thesis was to grow crystals and solve the structure of the hitherto uncrystallized cytochrome cL. The 1.6 Å X-ray crystal structure of cytochrome cL reported in this thesis was solved by molecular replacement; the search model was cytochrome c551i. As expected, the haem was covalently bonded to the protein through thioether bonds to Cys65 and Cys68 and the fifth ligand to the haem iron was provided by His69. Somewhat unexpectedly, the sixth ligand to the haem iron was provided by His112, and not Met109, which is homologous to Met101, the sixth ligand to the haem in cytochrome c551i. The absence of the N-terminal region of the protein in the electron density maps strongly suggested the protein has been truncated during crystallization, leading to an increase in the flexibility and solvent exposure of the loop region which contains Met109. This, along with the labile nature of the Met-Fe bond in cytochrome cL is the most-likely explanation for the bis-histidine haem iron coordination. The crystal structure also revealed that a calcium ion was bound close to the inner haem propionate; this region is most commonly occupied by an arginine or a histidine side chain in eukaryotic cytochromes c and bacterial cytochromes c2.