Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits.

Several mixed-function oxidation systems catalyze the inactivation of Escherichia coli glutamine synthetase. Inactivation involves modification of a single histidine residue in each enzyme subunit and makes the enzyme susceptible to proteolytic degradation. We show here that 10 key enzymes in metabolism are inactivated by a bacterial NADH oxidase and by an oxidase system comprised of NADPH, cytochrome P-450 reductase, and cytochrome P-450 isozyme 2 from rabbit liver microsomes. Most of the inactivatable enzymes require a divalent cation for activity and all but one (enolase) possess a nucleotide binding site. Glutamine synthetase, pyruvate kinase, and phosphoglycerate kinase are protected from inactivation by their substrates; substrate protection of other enzymes was not tested. We propose that inactivation involves mixed-function oxidization system-catalyzed synthesis of H2O2 and reduction of Fe(III) to Fe(II) followed by oxidation of enzyme-bound Fe(II) by H2O2 to generate oxygen radicals that attack a histidine (or other oxidizable amino acid) at the metal binding site of the enzyme. This is supported by the following: (i) most of the inactivation reactions are inhibited by EDTA and by catalase, (ii) both mixed-function oxidation systems reduce Fe(III), and (iii) H2O2 together with Fe(II) catalyzes nonenzymic inactivation of glutamine synthetase. In view of the fact that inactivation of glutamine synthetase makes it susceptible to proteolytic degradation, it is possible that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turn-over. In addition, the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed-function oxidation systems are known to accumulate as inactive forms during ageing.

https://www.pnas.org/content/pnas/80/6/1521.full.pdf

Inactivation of key metabolic enzymes by mixed-function oxidation reactions: Possible implication in protein turnover and ageing

The author describes studies that led to the resolution and reconstitution of the cytochrome P450 enzyme system in microsomal membranes. The review indicates how purification and characterization of the cytochromes led to rigorous evidence for multiple isoforms of the oxygenases with distinct chemical and physical properties and different but somewhat overlapping substrate specificities. Present knowledge of the individual steps in the P450 and reductase reaction cycles is summarized, including evidence for the generation of multiple functional oxidants that may contribute to the exceptional diversity of the reactions catalyzed.

Cytochrome P450: nature's most versatile biological catalyst.

On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction.

Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase.

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes.

Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450lm

Trypsinolysis of purified rabbit liver microsomal NADPH-cytochrome P-450 reductase yields a large, soluble fragment capable of reducing cytochrome c but not P-450 LM , and a small fragment or “tail peptide” which has been isolated and shown by its solubility and amino acid composition to be hydrophobic in nature. The large fragment is unable to bind to the cytochrome, as shown by gel exclusion chromatography and difference spectrophotometry. These and other experiments indicate that the hydrophobic tail peptide is required for formation of a functional complex between the reductase and P-450 LM in the reconstituted hydroxylation system.

John S. French

Papers

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes.

Role of a hydrophobic polypeptide in the N-terminal region of NADPH-cytochrome P-450 reductase in complex formation with P-450lm

Studies on the association of p-450 lm2 with nadph-cytochrome p-450 reductase and with tryptic peptides derived from the reductase