Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450

Arch Biochem Biophys. 1988 Mar;261(2):418-29. doi: 10.1016/0003-9861(88)90358-x.

Abstract

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the apparent Km of the reductase for cytochrome P-450b or cytochrome P-450c using either 7-ethoxycoumarin or benzphetamine as substrates. A 20-45% decrease in the Vmax was observed except for cytochrome P-450b with 7-ethoxycoumarin as substrate, where there was a 27% increase. Modification of carboxyl groups on the reductase with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, or taurine, respectively. The apparent Km for cytochrome P-450c or cytochrome P-450b was increased 1.3- to 5.2-fold in a reconstituted monooxygenase assay with 7-ethoxycoumarin or benzphetamine as substrate. There were varied effects on the Vmax. There was no significant change in the conformation of the reductase upon chemical modification with either acetic anhydride or EDC. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylation
  • Amino Acids
  • Cytochrome P-450 Enzyme System*
  • Electrochemistry
  • Electron Transport
  • Hydroxylation
  • Kinetics
  • NADPH-Ferrihemoprotein Reductase*
  • Oxidation-Reduction

Substances

  • Amino Acids
  • Cytochrome P-450 Enzyme System
  • NADPH-Ferrihemoprotein Reductase