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On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in brevibacterium sterolicum cholesterol oxidase.

ABSTRACT: The flavoprotein cholesterol oxidase from Brevibacterium sterolicum (BCO) possesses a narrow channel that links the active center containing the flavin to the outside solvent. This channel has been proposed to serve for the access of dioxygen; it contains at its "bottom" a Glu-Arg pair (Glu-475-Arg-477) that was found by crystallographic studies to exist in two forms named "open" and "closed," which in turn was suggested to constitute a gate functioning in the control of oxygen access. Most mutations of residues that flank the channel have minor effects on the oxygen reactivity. Mutations of Glu-311, however, cause a switch in the basic kinetic mechanism of the reaction of reduced BCO with dioxygen; wild-type BCO and most mutants show a saturation behavior with increasing oxygen concentration, whereas for Glu-311 mutants a linear dependence is found that is assumed to reflect a "simple" second order process. This is taken as support for the assumption that residue Glu-311 finely tunes the Glu-475-Arg-477 pair, forming a gate that functions in modulating the access/reactivity of dioxygen.

SUBMITTER: Piubelli L

PROVIDER: S-EPMC3259834 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in brevibacterium sterolicum cholesterol oxidase.

Piubelli Luciano L Pedotti Mattia M Molla Gianluca G Feindler-Boeckh Susanne S Ghisla Sandro S Pilone Mirella S MS Pollegioni Loredano L

The Journal of biological chemistry 20080709 36

The flavoprotein cholesterol oxidase from Brevibacterium sterolicum (BCO) possesses a narrow channel that links the active center containing the flavin to the outside solvent. This channel has been proposed to serve for the access of dioxygen; it contains at its "bottom" a Glu-Arg pair (Glu-475-Arg-477) that was found by crystallographic studies to exist in two forms named "open" and "closed," which in turn was suggested to constitute a gate functioning in the control of oxygen access. Most muta ...[more]

PMID: 18614534

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Project description:The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, determined at sub-angstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To elucidate the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or tunnel residue Phe359 or Gly347 to Trp or Asn reduces the catalytic efficiency of oxidation. The K mO 2 increases from 300 +/- 35 microM for the wild-type enzyme to 617 +/- 15 microM for the F359W mutant. The k cat for the F359W mutant-catalyzed reaction decreases 13-fold relative to that of the wild-type-catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively, is consistent with the cooperative kinetics. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

Dataset Information

On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in brevibacterium sterolicum cholesterol oxidase.

Publications

On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in brevibacterium sterolicum cholesterol oxidase.

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