Abstract
1. A methylene hydroxylase system from camphor induced Pseudomonas putida strain C1 cells has been separated into three fractions: a putidaredoxin reductase, putidaredoxin (an iron-sulfur protein), and a hydroxylase (shown to be a soluble cytochrome P-450). 2. This system of enzymes catalyzes the hydroxylation of methylene carbon 5 of camphor with reduced disphosphopyridine nucleotide as a primary electron donor and molecular oxygen as acceptor. 3. The redoxin serves as the electron carrier from the reduced pyridine nucleotide and reductase to the cytochrome P-450-substrate complex. 4. The coupling of the hydroxylase (P-450) to the reduced diphosphopyridine nucleotide dehydrogenase (reductase), and the putidaredoxin is demonstrated, and substrate is shown to be required for the over-all reaction.
Highlights
The system was separated into two protein fractions (A and B) and required a reduced pyridine nucleotide and molecular oxygen
The methylene hydroxylase of camphor-induced P. putida strain Cl was separated into the A and B fractions by chromatography on DEAE-cellulose according to the method of Cushman et al [2]
The addition of the third component, P-450 oAM, does not further increase the DPNH oxidation rate, but subsequent addition of substrate causes an immediate increase and leads to stoichiometric product formation according to the monoxygenase reaction shown in Scheme 1
Summary
The system was separated into two protein fractions (A and B) and required a reduced pyridine nucleotide and molecular oxygen. Fraction B, termed putidaredoxin’ to connote an iron-sulfur protein nature, was purified and characterized spectrally and catalytically by Cushman, Tsai, and Gunsalus [2].
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