Effects of Interdomain-Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidas

• Published in: Biochemistry (Easton) 2010-02, Vol.49 (6), p.1290
• Main Authors: Johnson-Winters, Kayunta, Nordstrom, Anna R., Emesh, Safia, Astashkin, Andrei V., Rajapakshe, Asha, Berry, Robert, Tollin, Gordon, Enemark, John H.
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9020296

Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b -type heme and two intermolecular one-electron steps to exogenous cytochrome c . In the crystal structure of chicken SO (Kisker et al., Cell , 1997, 91, 973–983), which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 Å, and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into closer proximity have been proposed (Feng et al., Biochemistry , 2003, 41, 5816–21) to explain the relatively rapid IET kinetics, which are much faster than theoretically predicted from the crystal structure. In order to explore the proposed role(s) of the tether in facilitating this conformational change, both its length and flexibility were altered in HSO by site-specific mutagenesis and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernable systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a three-fold decrease in the IET rate constant. Deletions of non-conserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steady-state kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a five-fold increase in the sulfite K m value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these Proline to Alanine and deletion mutants are identical to those of wild type HSO, indicating no significant change in the Mo active site geometry.