PROLYL-4-HYDROXYLASE OXYGEN ACTIVATION AND ASCORBATE PROTECTION

Open Access
Author:
Chekan, Jonathan Rodi
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Carsten Krebs, Thesis Supervisor
  • Joseph M Bollinger Jr., Thesis Supervisor
  • Sarah Ellen Ades, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • Ascorbate
  • Vitamin C
  • P4H
  • Prolyl-4-Hydroxylase
  • Reduction
  • Dioxygenase
Abstract:
It has long been known that ascorbate is necessary to the human diet for the proper formation of collagen. Without ascorbate, the enzyme prolyl-4-hydroxylase becomes inactivated and is no longer able to form the 4-hydroxyproline residues needed for stable collagen. However, the exact mechanism of inactivation has remained unknown. Using a P4H from PBCV-1, it was possible to determine enzymatic interactions with ascorbate. The results of this study demonstrate that ascorbate is able to reduce both singly and doubly oxidized iron species. By a one electron reaction, ascorbate reduces the inactive P4H•Fe(III) complex that forms by the adventitious oxidation of P4H•Fe(II). Alternatively, ascorbate can reduce the P4H•Fe(IV) intermediate. Presumably, the P4H•Fe(IV) is reduced by two single electron reduction reactions to P4H•Fe(III) then P4H•Fe(II). The reduction of P4H•Fe(IV) is of particular significance because P4H self-hydroxylation can occur when no substrate is present, a process called the “untriggered” reaction. By reducing the extremely reactive P4H•Fe(IV) intermediate that may form during the untriggered reaction, ascorbate is able to prevent enzymatic self hydroxylation and therefore inactivation. This study also sought to elucidate binding interactions between ascorbate, αKG, and P4H. It was shown that ascorbate and αKG directly compete with each other for binding to P4H.