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Probing the role of a conserved salt bridge in the intramolecular electron transfer kinetics of human sulfite oxidase

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Abstract

Sulfite oxidase (SO) is a vital metabolic enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed mechanism of this molybdenum cofactor dependent enzyme involves two one-electron intramolecular electron transfer (IET) steps from the molybdenum center to the iron of the b 5-type heme and two one-electron intermolecular electron transfer steps from the heme to cytochrome c. This work focuses on how the electrostatic interaction between two conserved amino acid residues, R472 and D342, in human SO (hSO) affects catalysis. The hSO variants R472M, R472Q, R472K, R472D, and D342K were created to probe the effect of the position of the salt bridge charges, along with the interaction between these two residues. With the exception of R472K, these variants all showed a significant decrease in their IET rate constants, k et, relative to wild-type hSO, indicating that the salt bridge between residues 472 and 342 is important for rapid IET. Surprisingly, however, except for R472K and R472D, all of the variants show k cat values higher than their corresponding k et values. The turnover number for R472D is about the same as k et, which suggests that the change in this variant is rate-limiting in catalysis. Direct spectroelectrochemical determination of the Fe(III/II) reduction potentials of the heme and calculation of the Mo(VI/V) potentials revealed that all of the variants affected the redox potentials of both metal centers, probably due to changes in their environments. Thus, the position of the positive charge of R472 and that of the negative charge of D342 are both important in hSO, and changing either the position or the nature of these charges perturbs IET and catalysis.

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Abbreviations

cSO:

Chicken sulfite oxidase

cyt c :

Cytochrome c

dRF:

5-Deazariboflavin

hSO:

Human sulfite oxidase

IET:

Intramolecular electron transfer

k et :

Electron transfer rate constant

K eq :

Equilibrium constant for intramolecular electron transfer

k f and k r :

Microscopic rate constants for the forward and reverse directions, respectively, of intramolecular electron transfer

Moco:

Molybdopterin cofactor

SO:

Sulfite oxidase

wt:

Wild type

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Acknowledgments

This research was supported by NIH grant GM-037773 (to J.H.E) and a Ruth L. Kirchstein–NIH Fellowship 1F32GM082136-01 (to K.J.-W); A.C.D. was a participant in the Undergraduate Biology Research Program and was supported in part by a grant to the University of Arizona from the Howard Hughes Medical Institute (52003749). We thank K.V. Rajagopalan for providing the pTG918 plasmid containing the hSO gene used to prepare the recombinant hSO enzyme. We thank F. Ann Walker for the use of equipment.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, 76019, USA

    Kayunta Johnson-Winters

  2. Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ, 85721, USA

    Amanda C. Davis, Anna R. Arnold, Robert E. Berry, Gordon Tollin & John H. Enemark

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  1. Kayunta Johnson-Winters
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  2. Amanda C. Davis
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  3. Anna R. Arnold
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Correspondence to Kayunta Johnson-Winters.

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Johnson-Winters, K., Davis, A.C., Arnold, A.R. et al. Probing the role of a conserved salt bridge in the intramolecular electron transfer kinetics of human sulfite oxidase. J Biol Inorg Chem 18, 645–653 (2013). https://doi.org/10.1007/s00775-013-1010-8

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