Erman, James E.
Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
Peroxidase||Cytochrome c||Chemistry, Physical and theoretical
The reaction mechanism of cytochrome c peroxidase (CcP) was outlined by Poulos and Kraut many years ago, but the role and interaction of residues in the distal binding pocket are poorly understood. His-52 clearly acts as a base catalyst in CcP's reaction with H2O2, but other highly-conserved residues in the binding pocket of peroxidases have important, but as yet undefined, roles. For instance, metmyoglobin has many of the structural features of CcP, including a histidine on the distal side of the heme iron, yet it is a poor peroxidase. We created multiple mutants of CcP in which His-52 is replaced to investigate the role of base strength versus hydrogen bonding in the formation of Compound I. We also created mutants substituting histidine for Trp-51 in order to test whether the distal histidine of metmyoglobin (4.3 Å from the heme iron, versus 5.6 Å for CcP) is too close to react rapidly with hydrogen peroxide. The mutants were tested with hydrogen peroxide at pH 4, 6 and 8 and with cyanide at pH 6. There is strong correlation between rate of cyanide binding and rate of Compound I formation, indicating that cyanide binding is a useful model to study the formation of Compound I. The cyanide data also verified that, even in mutants lacking a base catalyst at position 52, the association of hydrogen peroxide and the heme iron is the rate-limiting step. Base strength correlates well with the rate of Compound I formation, but residues that could only hydrogen-bond to the reaction intermediate had little effect on the apparent bimolecular rate constant. Moving histidine to position 51 and replacing His-52 with leucine or tryptophan decreases the rate of reaction with H₂O₂ relative to wild type by 2.5 to 3.5 orders of magnitude, but the rate is still 2—-3 orders of magnitude faster than the rate for soluble heme analogs or microperoxidase. Other steric considerations also affect the rate. Replacing His-52 with lysine (H52K) produces a mutant with an H₂O₂ reaction rate 1/50th the rate of wild-type CcP. CcP(H52K) apparently has many conformers at neutral pH, and a group controlling the rate of reaction with hydrogen peroxide, presumed to be lysine, exhibits a pKₐ of 6.3, four orders of magnitude lower than the pKₐ of free lysine. This dramatic change apparently results from the two positive charges in the binding pocket, and may have the same effect on hydrogen peroxide, explaining why CcP(H52L), with no base at position 52, and CcP(H52K) at pH 4, where the lysine is protonated, still react faster with H₂O₂ than model compounds.
Foshay, Miriam C., "A study of structure/function relationships in cytochrome c peroxidase : base catalysis and hydrogen-bonding in the formation of compound I" (2002). Graduate Research Theses & Dissertations. 681.
xxxii, 493 pages
Northern Illinois University
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