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 cytochrome c-550 from the nonphotosynthetic bacteria Paraccocus versutus has been solved by X-ray crystallography to 1.90 A resolution, and reveals a high structural homology to other bacterial cytochromes c(2). 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 1.95 and 1.55 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-conditions 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.
Worrall
Jonathan A R
JA
Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, Leiden, the Netherlands.
van Roon
Anne-Marie M
AM
Ubbink
Marcellus
M
Canters
Gerard W
GW
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry; D008024:Ligands; D008239:Lysine / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010230:Paracoccus / Q000737:chemistry*; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D008024:Ligands; D006418:Heme; C031732:cytochrome C-550; D008715:Methionine; D010544:Peroxidases; D008239:Lysine