Inactivation of the heme degrading enzyme IsdI by an active site substitution that diminishes heme ruffling.
IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (-96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (-89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada.
Georgia
G
Ukpabi
Shin-ichi J
SJ
Takayama
A Grant
AG
Mauk
Michael E P
ME
Murphy
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000187:drug effects*
D001426:Bacterial Proteins; D006418:Heme; C497164:IsdI protein, Staphylococcus aureus; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C482079:IsdG protein, Staphylococcus aureus