Cytochromes c(555) from the hyperthermophilic bacterium Aquifex aeolicus. 2. Heterologous production of soluble cytochrome c(555)(s) and investigation of the role of methionine residues

The cycB2 gene encoding the soluble cytoehrome c(555)(s) from Aquifer aeoli cus, an hyperthermophilic organism, has been cloned and expressed using Esc herichia coli as the host organism. The cytochrome was successfully produce d in the periplasm of an E. coli strain coexpressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. Comparison of native and r ecombinant cytochrome C-555(s) shows that both proteins are indistinguishab le in terms of spectroscopic and physicochemical properties. Since two diff erent methionine residues are present in the sequence stretch usually provi ding 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 me thionine by a histidine residue. Characterization of both mutants, M78H and M84H cytochromes c(555)(s), using biochemical and biophysical techniques h as been carried out. The M84H mutant exhibits spectral features identical t o those of native cytochrome. Its redox midpoint potential is decreased by 40 mV. By contrast, substitution of methionine 78 by a histidine residue st rongly alters the structural and physicochemical properties of the molecule which exhibits characteristics of HiS/HiS iron coordination type rather th an His/Met. These results allow us to identify methionine 78 as the sixth l igand of cytochrome c(555)(s) heme iron. Preliminary results on the thermos tability of the native and mutant cytochromes c(555) are also reported.