Universität zu Köln

Eh, Malin, Kaczmarek, Alexander Tobias, Schwarz, Guenter ORCID: 0000-0002-2118-9338 and Bender, Daniel ORCID: 0000-0002-2975-4186 (2022). Molecular mechanism of intramolecular electron transfer in dimeric sulfite oxidase. J. Biol. Chem., 298 (3). AMSTERDAM: ELSEVIER. ISSN 1083-351X

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Abstract

Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo(IV)or Mo(V) follow this path.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Eh, Malin UNSPECIFIED UNSPECIFIED UNSPECIFIED Kaczmarek, Alexander Tobias UNSPECIFIED UNSPECIFIED UNSPECIFIED Schwarz, Guenter UNSPECIFIED orcid.org/0000-0002-2118-9338 UNSPECIFIED Bender, Daniel UNSPECIFIED orcid.org/0000-0002-2975-4186 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-668044
DOI:	10.1016/j.jbc.2022.101668
Journal or Publication Title:	J. Biol. Chem.
Volume:	298
Number:	3
Date:	2022
Publisher:	ELSEVIER
Place of Publication:	AMSTERDAM
ISSN:	1083-351X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MOLYBDENUM; NITRITE; MITOCHONDRIA; DEFICIENCY; CATALYSIS; COFACTOR; IMPORT; G473D Multiple languages Biochemistry & Molecular Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/66804