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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Oct;71(10):3906–3910. doi: 10.1073/pnas.71.10.3906

A Role of the Putidaredoxin COOH-terminus in P-450cam (Cytochrome m) Hydroxylations*

S G Sligar 1,2, P G Debrunner 1,2, J D Lipscomb 1,2, M J Namtvedt 1,2, I C Gunsalus 1,2
PMCID: PMC434294  PMID: 4530269

Abstract

Methylene hydroxylation by cytochrome P-450cam (cytochrome m) can be resolved into four distinct steps: substrate addition, mo → mos; reduction, mos → mrs; dioxygen addition, mrs → mO2rs; followed by a second putidaredoxin (Pseudomonas putida ferredoxin)-mediated reduction and product formation. The isolated ferrous oxy-substrate complex exhibits first-order decay kinetics with the relatively slow rate constant of k [unk] 0.01 sec-1, at 25°, without product release. Putidaredoxin addition accelerates the decomposition with second-order kinetics, k [unk] 51,000 M-1 sec-1, and initiation of product formation. Cytochrome m forms a complex with putidaredoxin with dissociation constant of KD = 3 μM. In the complete three-protein hydroxylase system, consisting of cytochrome m, putidaredoxin, and the reductase (a DPNH-specific flavo-protein), camphor hydroxylation occurs with a stoichiometry of 1 mole each of DPNH and O2 used per mole of product formed; the KM for putidaredoxin is about 4.2 μM.

Putidaredoxin, on treatment with carboxypeptidase A, loses one molecule each of tryptophan and glutamine sequentially from the carboxy terminus to expose a terminal arginine. The tryptophan-free product has been separated from native putidaredoxin and other impurities, and retains the visible and electron paramagnetic resonance spectra and the redox potential of the active center of native putidaredoxin. This modified redoxin binds less tightly to cytochrome m, KD [unk] 150 μM, and is 50 times less effective in stimulation of the mO2rs decay rate. A similar decrease in specific activity is observed in the complete hydroxylase system.

Keywords: monooxygenase activity, protein modification, iron-sulfur protein-cytochrome complex, fluorescence, cytochrome P-450

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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