A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s

Dipanwita Batabyal, Ariel Lewis-Ballester, Syun-Ru Yeh, Thomas L. Poulos

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two-iron-sulfur ferredoxin. The closest homologue to P450cam, CYP101D1, is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor to give the same single 5-exo-hydroxy camphor product. However, CYP101D1 does not strictly require its own ferredoxin (Arx) for activity because Pdx can support CYP101D1 catalysis but Arx cannot support P450cam catalysis. We have further examined the differences between these two P450s by determining the effect of spin equilibrium, redox properties, and stability of oxygen complexes. We find that Arx shifts the spin state equilibrium toward high-spin, which is the opposite of the effect of Pdx on P450cam. In both P450s, redox partner binding destabilizes the oxy-P450 complex but this effect is much weaker with CYP101D1. In addition, resonance Raman data show that structural perturbations observed in P450cam upon addition of Pdx are absent in CYP101D1. These data indicate that Arx does not play the same effector role in catalysis as Pdx does with P450cam. The most relevant structural difference between these two P450s centers on a catalytically important Asp residue required for proton-coupled electron transfer. We postulate that with P450cam larger Pdx-assisted motions are required to free this Asp for catalysis while the smaller number of restrictions in CYP101D1 precludes the need for redox partner-assisted structural changes.

Original languageEnglish (US)
Pages (from-to)6517-6523
Number of pages7
JournalBiochemistry
Volume55
Issue number47
DOIs
StatePublished - Nov 29 2016

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Camphor 5-Monooxygenase
Oxidation-Reduction
Camphor
Catalysis
Ferredoxins
Viperidae
Catalyst supports
Cytochromes
Mixed Function Oxygenases
Sulfur
putidaredoxin
Protons
Catalyst activity
Iron
Electrons
Oxygen

ASJC Scopus subject areas

  • Biochemistry

Cite this

A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s. / Batabyal, Dipanwita; Lewis-Ballester, Ariel; Yeh, Syun-Ru; Poulos, Thomas L.

In: Biochemistry, Vol. 55, No. 47, 29.11.2016, p. 6517-6523.

Research output: Contribution to journalArticle

Batabyal, Dipanwita ; Lewis-Ballester, Ariel ; Yeh, Syun-Ru ; Poulos, Thomas L. / A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s. In: Biochemistry. 2016 ; Vol. 55, No. 47. pp. 6517-6523.
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AB - The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two-iron-sulfur ferredoxin. The closest homologue to P450cam, CYP101D1, is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor to give the same single 5-exo-hydroxy camphor product. However, CYP101D1 does not strictly require its own ferredoxin (Arx) for activity because Pdx can support CYP101D1 catalysis but Arx cannot support P450cam catalysis. We have further examined the differences between these two P450s by determining the effect of spin equilibrium, redox properties, and stability of oxygen complexes. We find that Arx shifts the spin state equilibrium toward high-spin, which is the opposite of the effect of Pdx on P450cam. In both P450s, redox partner binding destabilizes the oxy-P450 complex but this effect is much weaker with CYP101D1. In addition, resonance Raman data show that structural perturbations observed in P450cam upon addition of Pdx are absent in CYP101D1. These data indicate that Arx does not play the same effector role in catalysis as Pdx does with P450cam. The most relevant structural difference between these two P450s centers on a catalytically important Asp residue required for proton-coupled electron transfer. We postulate that with P450cam larger Pdx-assisted motions are required to free this Asp for catalysis while the smaller number of restrictions in CYP101D1 precludes the need for redox partner-assisted structural changes.

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