Abstract
We have solved the x-ray structures of the binary horseradish peroxidase C-ferulic acid complex and the ternary horseradish peroxidase C-cyanide-ferulic acid complex to 2.0 and 1.45 A, respectively. Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and is an in vivo substrate for plant peroxidases. The x-ray structures demonstrate the flexibility and dynamic character of the aromatic donor binding site in horseradish peroxidase and emphasize the role of the distal arginine (Arg(38)) in both substrate oxidation and ligand binding. Arg(38) hydrogen bonds to bound cyanide, thereby contributing to the stabilization of the horseradish peroxidase-cyanide complex and suggesting that the distal arginine will be able to contribute with a similar interaction during stabilization of a bound peroxy transition state and subsequent O-O bond cleavage. The catalytic arginine is additionally engaged in an extensive hydrogen bonding network, which also includes the catalytic distal histidine, a water molecule and Pro(139), a proline residue conserved within the plant peroxidase superfamily. Based on the observed hydrogen bonding network and previous spectroscopic and kinetic work, a general mechanism of peroxidase substrate oxidation is proposed.
Highlights
Compound IHRPC[(Fe(IV)¢O)Porph1⁄7Ϫ]1⁄7ϩ ϩ AH 3 HRPC[(Fe(IV)¢O)Porph2Ϫ] ϩ Hϩ ϩ A1⁄7 Compound II
We have solved the x-ray structures of the binary horseradish peroxidase C-ferulic acid complex and the ternary horseradish peroxidase C-cyanide-ferulic acid complex to 2.0 and 1.45 Å, respectively
Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and is an in vivo substrate for plant peroxidases
Summary
HRPC[(Fe(IV)¢O)Porph1⁄7Ϫ]1⁄7ϩ ϩ AH 3 HRPC[(Fe(IV)¢O)Porph2Ϫ] ϩ Hϩ ϩ A1⁄7 Compound II. The oxidation of native enzyme by H2O2 (Reaction 1) is well understood, and numerous experiments [17] have confirmed the general catalytic mechanism for this step first proposed by Poulos and Kraut [18]. The oxidation of phenolic substrates (Reactions 2 and 3) has been less well understood, but a histidine [19] (His in HRPC) and an arginine [20] (Arg in HRPC) have been shown to contribute significantly to enhance the rate of substrate oxidation. There is, no direct correlation between the Kd of a substrate and its reactivity toward compounds I and II. In general the rates of reaction of compound I with substrates correlate well with the driving force for the reaction [25], the HOMO/LUMO energy levels of substrate molecules [26], or
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