Electron transfer between cofactors in protein domains linked by a flexible tether

Kawatsu, Tsutomu; Beratan, David N.

doi:10.1016/j.chemphys.2006.01.020

Cited by 16 publications

(32 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, for SO it is possible that k et is the result of many averaged conformations of SO, as well as the overall driving force and reorganization energy (62). Finally, this study provides experimental data for testing computational models for electron transfer in a two domain enzyme (45). However, such calculations are beyond the scope of the present work.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

View full text Add to dashboard Cite

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 ...

show abstract

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

View full text Add to dashboard Cite

show abstract

“…Prior to 2010 there were no experimental data from site-specific mutants to investigate the role of conformational changes of the tether that had been proposed from the effects of viscosity on IET (70) and from theoretical calculations (71). Recently, the proposed role(s) of the tether in facilitating conformational change and reactivity have been explored by altering both the length and flexibility of the tether in HSO by site-specific mutagenesis, and the reactivities of the resulting variants were studied using laser flash photolysis and steady-state kinetics assays (79).…”

Section: Intramolecular Electron Transfer (Iet) Kinetic Studies In Sumentioning

confidence: 99%

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

2010

View full text Add to dashboard Cite

Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor dependent enzymes that are found in plants, animals and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO catalyzes the oxidation of toxic sulfite to sulfate as the final step in the catabolism of the sulfur-containing amino acids, methionine and cysteine. In humans, sulfite oxidase deficiency is an inherited recessive disorder that produces severe neonatal neurological problems that lead to early death. Plant SO (PSO) also plays an important role in sulfite detoxification, but in addition serves as an intermediate enzyme in the assimilatory reduction of sulfate. In vertebrates the proposed catalytic mechanism of SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme. A similar mechanism is proposed for SDH, involving its molybdenum cofactor and c-type heme. However, PSO, which lacks an integral heme cofactor, uses molecular oxygen as its electron acceptor. Here we review recent results for SOEs from kinetic measurements, computational studies, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements, and site-directed mutagenesis on active site residues of SOEs and of the flexible polypepetide tether that connects the heme and molybdenum domains of human SO. Rapid-kinetic studies of PSO are also discussed.

show abstract

“…A recent interesting simulation study suggests that the tether length may control the transition between the electron tunneling and diffusion-limited regimes in the IET reaction [91].…”

mentioning

confidence: 99%

Sulfite-oxidizing enzymes

Kappler

Enemark

2014

J Biol Inorg Chem

View full text Add to dashboard Cite

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.

show abstract

Electron transfer between cofactors in protein domains linked by a flexible tether

Cited by 16 publications

References 41 publications

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

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

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

Sulfite-oxidizing enzymes

Contact Info

Product

Resources

About