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Abstract

The cytochrome c domain of subunit II from the Rhodothermus marinus caa₃ HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 Å by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R_cryst and R_free values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel β-strands forming a small β-sheet, an interesting variation of the classical all α-helical cytochrome c fold. This modification appears to be common to all known caa₃-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa₃-type terminal oxidase. The R. marinus caa₃ uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.