CD and MCD of CytC3 and taurine dioxygenase: role of the facial triad in alpha-KG-dependent oxygenases.
The alpha-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require FeII, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the FeII active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An FeII methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of FeII for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the FeII/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.
Neidig
Michael L
ML
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Brown
Christina D
CD
Light
Kenneth M
KM
Fujimori
Danica Galonić
DG
Nolan
Elizabeth M
EM
Price
John C
JC
Barr
Eric W
EW
Bollinger
J Martin
JM
Krebs
Carsten
C
Walsh
Christopher T
CT
Solomon
Edward I
EI
D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D054879:Halogenation; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D006900:Hydroxylation; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D019159:Nonheme Iron Proteins / Q000737:chemistry / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D002264:Carboxylic Acids; D007656:Ketoglutaric Acids; D008024:Ligands; D019159:Nonheme Iron Proteins; D006639:Histidine; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C066046:clavaminate synthase; C433064:taurine-alpha-ketoglutarate dioxygenase