Effects of large-scale amino acid substitution in the polypeptide tether connecting the heme and molybdenum domains on catalysis in human sulfite oxidase

Abstract

Sulfite oxidase (SO) is a molybdenum-cofactor-dependent enzyme that catalyzes the oxidation of sulfite to sulfate, the final step in the catabolism of the sulfur-containing amino acids, cysteine and methionine. The catalytic mechanism of vertebrate SO involves intramolecular electron transfer (IET) from molybdenum to the integral b-type heme of SO and then to exogenous cytochrome c. However, the crystal structure of chicken sulfite oxidase (CSO) has shown that there is a 32 Å distance between the Fe and Mo atoms of the respective heme and molybdenum domains, which are connected by a flexible polypeptide tether. This distance is too long to be consistent with the measured IET rates. Previous studies have shown that IET is viscosity dependent (Feng et al., Biochemistry, 2002, 41, 5816) and also dependent upon the flexibility and length of the tether (Johnson-Winters et al., Biochemistry, 2010, 49, 1290). Since IET in CSO is more rapid than in human sulfite oxidase (HSO) (Feng et al., Biochemistry, 2003, 42, 12235) the tether sequence of HSO has been mutated into that of CSO, and the resultant chimeric HSO enzyme investigated by laser flash photolysis and steady-state kinetics in order to study the specificity of the tether sequence of SO on the kinetic properties. Surprisingly, the IET kinetics of the chimeric HSO protein with the CSO tether sequence are slower than wildtype HSO. This observation raises the possibility that the composition of the non-conserved tether sequence of animal SOs may be optimized for individual species.

Determination of the Distance between the Mo(V) and Fe(III) Heme Centers of Wild Type Human Sulfite Oxidase by Pulsed EPR Spectroscopy

“…This Pro to Ala mutation would be expected to increase the flexibility of the tether and hence the orientations accessible to the heme domain. Replacement of the tether sequence of hSO with that of cSO unexpectedly gave slower IET, even though wt cSO has faster IET than wt hSO …”

Elucidating the Catalytic Mechanism of Sulfite Oxidizing Enzymes Using Structural, Spectroscopic, and Kinetic Analyses

“…The expectation was that this chimeric HSO protein would show IET kinetics that were faster than those of wt HSO and more similar to those of CSO. Surprisingly, however, IET for the chimeric HSO was actually slower than that for wt HSO . This result suggests that the composition of the nonconserved tether sequence of animal SOs may have become optimized for individual species.…”

Kinetic and Thermodynamic Effects of Mutations of Human Sulfite Oxidase

“…The effect of change in the flexibility has been explored by replacing two prolines, (P105 and P111) by alanines in hSO [4–6]. The P105 residue is adjacent to the heme domain, and the P105A mutant would presumably have increased tether flexibility.…”

“…The effect of the tether length has been explored by shortening the tether by deleting three (KVA), four (KVAT), and five (KVATV) non-conserved amino acid resides from the tether [5, 6]. The largest decrease in the IET rate was for the five amino acid deletion mutant, and IET became the rate-limiting step in catalysis (Table 1).…”