tailieunhanh - Báo cáo khoa học: The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c-550 from Paracoccus versutus assessed by X-ray crystallography and unfolding

The structure of cytochromec-550 from the nonphotosynthetic bacteria Paraccocus versutushas been solved by X-ray crystallography to A˚ resolution, and reveals a high structural homology to other bacterial cyto-chromesc2. The effect of replacing the axial heme-iron methionine ligand with a lysine residue on protein structure and unfolding has been assessed using the M100K variant. | ềFEBS Journal The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c-550 from Paracoccus versutus assessed by X-ray crystallography and unfolding Jonathan A. R. Worrall Anne-Marie M. van Roon Marcellus Ubbink and Gerard W. Canters Leiden Institute of Chemistry Leiden University Gorlaeus Laboratories Leiden the Netherlands Keywords axial ligand cytochrome c unfolding peroxidase activity X-ray crystallography Correspondence G. W. Canters Leiden Institute of Chemistry Leiden University Gorlaeus Laboratories PO Box9502 2300 RA Leiden the Netherlands Fax 31 71 527 4349 Tel 31 71 527 4256 E-mail canters@ Received 14 January 2005 revised 9 March 2005 accepted 15 March 2005 doi The structure of cytochrome c-550 from the nonphotosynthetic bacteria Paraccocus versutus has been solved by X-ray crystallography to A resolution and reveals a high structural homology to other bacterial cytochromes c2. The effect of replacing the axial heme-iron methionine ligand with a lysine residue on protein structure and unfolding has been assessed using the M100K variant. From X-ray structures at and A resolution it became clear that the amino group of the lysine side chain coordinates to the heme-iron. Structural differences compared to the wild-type protein are confined to the lysine ligand loop connecting helices four and five. In the heme cavity an additional water molecule is found which participates in an H-bonding interaction with the lysine ligand. Under cryo-con-ditions extra electron density in the lysine ligand loop is revealed leading to residues K97 to T101 being modeled with a double main-chain conformation. Upon unfolding dissociation of the lysine ligand from the heme-iron is shown to be pH dependent with NMR data consistent with the occurrence of a ligand exchange mechanism similar to that seen for the wild-type protein. The Gram-negative nonphotosynthetic bacterium .