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

Abstract

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 steadystate 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.Sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate, using oxidized ferricytochrome c (cyt c ox ) as the physiological electron acceptor (eq. 1) (1-4). This reaction is biologically essential, serving as the final step in the catabolism of sulfur containing amino acids, methionine and cysteine, and as a detoxification mechanism for sulfite. † This research was supported by NIH Grant GM-037773 (to JHE); Ruth L. Kirchstein-NIH Fellowship 1F32GM082136-01 (to KJW) *To whom correspondence should be addressed. J.H.E.: jenemark@u.arizona.edu; phone, (520) 621-2245; fax, (520) 626-8065. G.T.:, gtollin@u.arizona.edu; phone, (520) 621-3447; fax, (520) 621-9288. Supporting Information Available: Primer design; iron to molybdenum ratios determined using inductively coupled plasma; and laser flash photolysis results for proline to alanine ...

Sulfite Oxidase Catalyzes Single-Electron Transfer at Molybdenum Domain to Reduce Nitrite to Nitric Oxide

“…The fact that the apo-heme domain also inhibits nitrite reduction, to some extent, suggests an important conformational component in the access of nitrite to sulfite-reduced SO. There is a flexible hinge (14 residues in human SO) that tethers N-terminal heme domain to Mo-domain (21). Previous studies have shown that this hinge controls domain motion during the catalytic cycle of SO, which adopts various conformations during intramolecular electron transfer (IET) and catalysis (49).…”

Kinetic results for mutations of conserved residues H304 and R309 of human sulfite oxidase point to mechanistic complexities

“…11) using the nonlinear-least-squares fitting algorithm in the software program Origin © : $$E_{app}=E^0+2.303(\mathrm{RT}∕\mathrm{nF}){\log }_{10}(\left[\mathrm{Ox}\right]∕\left[\mathrm{Red}\right])$$ where E app is the applied potential, E 0 is the midpoint potential determined from these data, and [Ox] and [Red] are, respectively, the concentrations of the Fe(III) and Fe(II) states of the b 5 heme of hSO. For a previous application of this method to hSO see Figure 4 of ref 17.…”

“…These mutations include: deletion of tether residues (17); mutation of the aromatic residues around both the heme and Mo domains (18, 19); mutation of conserved active site residues (20-22); and producing some of the fatal mutations (22-26) such as R160Q and K322R. Although many of these mutants have provided much insight into the structural and kinetic characteristics of hSO, some have shown unexplained or even paradoxical results.…”

“…For example, mutation of H337 and W338 on the surface of the Mo domain influence the potential of the heme domain, despite the large distance between these residues and the heme in the crystal structure (18). Mutations such as K322R, W338A, H337R, and ΔKVATV, show IET rates that are much less than wt hSO and sometimes less their own k cat values, while k cat in many cases has increased significantly as compared to wt (17, 18, 25). These results illustrate the complexity of hSO and indicate that the roles of conformational change and other factors in the catalytic cycle and IET reactions of hSO and their relationship to isolated sulfite oxidase deficiency are yet to be understood.…”

Structural Evidence for the Functional Importance of the Heme Domain Mobility in Flavocytochrome b2

“…50 In the family of cytochrome b 5 homologues, microsomal cytochrome b 5 itself is connected to its transmembrane segment by a flexible linker: the latter enables cytochrome b 5 to interact alternatively with its reductases and its diverse electron acceptors, all of them attached to the endoplasmic reticulum. 33,51 Sulfite oxidase is the system with most similarity to Fcb2, in that the b 5 -like heme domain is covalently attached to its reductase, a molybdopterin domain, and transfers electrons to cytochrome c. In this case, the crystal structure of the chicken liver enzyme is not considered as that of an electron transfercompetent complex, and the flexible peptide loop must allow the orientation changes necessary for shuttling electrons between the molybdopterin domain and cytochrome c. [52][53][54] In all structural and modeling studies with cytochrome b 5 mentioned above, the surface that interacts with electron acceptors is that around the exposed heme edge. Thus far, Fcb2 is the only system for which both crystallographic and functional details exist concerning the electron transfer-competent interaction of a b 5 -like cytochrome with its reductase, an interaction that again implicates the area around the exposed heme edge.…”