Cytochromes c sub(555) from the Hyperthermophilic Bacterium Aquifex aeolicus. 2. Heterologous Production of Soluble Cytochrome c sub(555) super(s) and Investigation of the Role of Methionine Residues

The cycB2 gene encoding the soluble cytochrome c sub(555) super(s) from Aquifex aeolicus, an hyperthermophilic organism, has been cloned and expressed using Escherichia coli as the host organism. The cytochrome was successfully produced in the periplasm of an E. coli strain coexpressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. Comparison of native and recombinant cytochrome c sub(555) super(s) shows that both proteins are indistinguishable in terms of spectroscopic and physicochemical properties. Since two different methionine residues are present in the sequence stretch usually providing the sixth ligand to the heme iron, site-directed mutagenesis has been performed in order to identify the methionine serving as the axial ligand. Two single mutations were introduced, leading to the replacement of each methionine by a histidine residue. Characterization of both mutants, M78H and M84H cytochromes c sub(555) super(s), using biochemical and biophysical techniques has been carried out. The M84H mutant exhibits spectral features identical to those of native cytochrome. Its redox midpoint potential is decreased by 40 mV. By contrast, substitution of methionine 78 by a histidine residue strongly alters the structural and physicochemical properties of the molecule which exhibits characteristics of His/His iron coordination type rather than His/Met. These results allow us to identify methionine 78 as the sixth ligand of cytochrome c sub(555) super(s) heme iron. Preliminary results on the thermostability of the native and mutant cytochromes c sub(555) are also reported.