Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis

doi:10.1073/pnas.0510482103

Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis

^†Biophysics Research Division and
Departments of ^‡Physics,
^§Electrical Engineering and Computer Science, and
^¶Biological Chemistry, University of Michigan, Ann Arbor, MI 48109

Edited by Gordon G. Hammes, Duke University Medical Center, Durham, NC, and approved January 25, 2006 (received for review December 5, 2005)

Abstract

Subunit activity and cooperativity of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA) from Lactococcus lactis, were characterized by employing single-molecule spectroscopy to follow the turnover kinetics of individual DHODA molecules, eliminating ensemble averaging. Because the enzyme-bound FMN is fluorescent in its oxidized state but not when reduced, a single DHODA molecule exhibits stepwise fluorescence changes during turnover, providing a signal to determine reaction kinetics and study cooperativity. Our results showed significant heterogeneity in the catalytic behaviors of individual dimer molecules, with only 40% interconverting between the three possible redox states: the fully fluorescent (both subunits oxidized), the half-fluorescent (one subunit oxidized and the other reduced), and the nonfluorescent (both subunits reduced). Forty percent of the single dimer traces showed turnovers between only the fully fluorescent and half-fluorescent states. The remaining 20% of the molecules interconverted only between the half-fluorescent state and the nonfluorescent state. Kinetic analysis revealed very similar reaction rates in both the reductive and oxidative half-reactions for different DHODA dimers. Our single-molecule data provide strong evidence for half-sites reactivity, in which only one subunit reacts at a time. The present study presents an effective way to explore the subunit catalytic activity and cooperativity of oligomeric enzymes by virtue of single-molecule fluorescence.

Footnotes

^‖To whom correspondence should be addressed. E-mail: brupalf{at}umich.edu
Author contributions: J.S., A.G., D.S., and B.A.P. designed research; J.S. performed research; J.S. and J.D. contributed new reagents/analytic tools; J.S., A.G., D.S., and B.A.P. analyzed data; and J.S., A.G., D.S., and B.A.P. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

DHO,

dihydroorotate;

DHODA,

DHO dehydrogenase A.