pubs.acs.org/JPCL
Three-Dimensional Structural Model of Chicken Liver Sulfite Oxidase in its Activated Form Tillmann Utesch and Maria Andrea Mroginski* Technische Universit€ at Berlin, Institut f€ ur Chemie, Max-Volmer- Laboratorium, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin, Germany
ABSTRACT Sulfite oxidase (SO) catalyzes the conversion of sulfite to sulfate in almost all living organisms. In vertebrates, the catalytic process involves a rapid intramolecular electron transfer (IET) step between the molybdenum cofactor in the central domain and the heme in the cytochrome b5 domain. The large distance between redox centers observed in the crystal structure disagrees with the fast IET rates measured experimentally. This conflict was explained by postulating a major rearrangement of the cytochrome b5 domain toward the molybdopterin cofactor. Using steered molecular dynamics and molecular dynamics simulations, we generated a stable 3D structural model for chicken liver SO (CSO) in the activated form characterized by a short electron donor-acceptor distance consistent with the enzymes' experimentally obtained electron transfer properties. IET rates for the active complex were estimated with the Pathway model. The good agreement between calculated and experimental IET rates supports our structural model for the active CSO. SECTION Molecular Structure, Quantum Chemistry, General Theory
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Pacheco and coworkers were able to explain the high IET rates in SO despite the large Moco-to-heme distance by introducing the idea of a very flexible cytochrome b5 domain.4 In their model, they proposed the motion of the negatively charged cytochrome b5 domain toward the cationic substrate-binding pocket to reduce the Mo-Fe distance and achieve fast IET rates obtained experimentally. In the proposed scheme, the flexible linker region connecting the two domains acts as a hinge and enables this rearrangement of the anionic cytochrome b5 domain toward the active site. This hypothesis was supported by the observation made from laser flash photolysis experiments of a decreased IET with increasing viscosity of the solution.6 Additionally, molecular dynamics (MD) simulations of CSO monomer performed by Pushie et al.7 support the idea of a mobile cytochrome b5 domain. In their work, they observed a highly mobile heme domain and estimated separation distances between the two redox centers varying from 27.4 to 56.9 Å. The effect of the tether flexibility on the IET reaction kinetics was investigated by Kawatsu and Beratan8 using a computational approach and very recently by Enemark and coworkers through laser flash photolysis and steady-state kinetics experiments.9 Despite the several experimental and theoretical attempts accomplished to unravel the structural properties of the SO in a catalytically active form characterized by a high IET rate and a
ulfite-oxidizing enzymes are found in prokaryotic and eukaryotic organisms.1 Vertebrate sulfite oxidase (SO) is a homodimeric enzyme that catalyzes the final step in the degradation of sulfur-containing amino acids by oxidation of sulfite to sulfate with 2 equiv of ferricytochrome c (cyt c) as the terminal electron acceptor1,2 SO3 2 - þ H2 O þ 2ðcyt cÞox SSO4 2 - þ 2Hþ þ 2e - þ 2ðcyt cÞred
During catalysis, the oxidized molybdenum cofactor (Moco) located in the central domain takes up two electrons, and the Mo(VI) is reduced to the Mo(IV) state. The received electrons are sequentially transferred from the Mo to the oxidized Fe of the heme group located in the N-terminal cytochrome b5 domain. In animals, the two domains are connected via a very flexible loop of 10 amino acids shown in the crystallographic structure of chicken liver sulfite oxidase (CSO)3 (Figure 1). This flexible loop is assumed to play an important role in the intramolecular electron transfer (IET) process because it facilitates the reorientation of the cytochrome b5 domain toward the Moco harboring domain.3,4 After reduction of the heme, the electrons are transferred further to the external electron acceptor ferricytochrome c. To date, the IET process is not completely understood. One of the most challenging questions is the discrepancy between high electron transfer (ET) rates measured in experiments5 and the large distance between the two redox centers, of ca. 32 Å, observed in the crystallographic structure of CSO.3 CSO has a high sequence similarity to human SO and is a typical representative for sulfite oxidases of higher vertebrates.
r 2010 American Chemical Society
Received Date: May 7, 2010 Accepted Date: June 25, 2010 Published on Web Date: July 01, 2010
2159
DOI: 10.1021/jz1005847 |J. Phys. Chem. Lett. 2010, 1, 2159–2164
pubs.acs.org/JPCL
with the Moco and dimerization domains, the rmsd of the cytochrome b5 suddenly increases to ∼2.0 Å. After removing the external forces from the system, we observed a quick reorganization of the cytochrome b5 domain, resulting in an increase in the Mo-Fe distance from ∼17.5 to >20 Å (Figure 2a). In the relaxation and equilibration periods, the rmsd of the cytochrome b5 domain remained steady around 1.85 Å, indicating a stable conformation without large structural changes (Figure 2b). Moreover, during the 14 ns simulation, the distance between the two redox partners fluctuated between 17 and 20 Å without increasing. This indicates that there is no repulsion between the cytochrome b5 and Moco domains. The root-mean-square fluctuations (rmsf's) calculated from the MD simulation for all CR of the SO monomer show a prominent peak in the linker region connecting the cytochrome b5 domain to the Moco domain. The fluctuations in the loop are nearly twice as large as those in the cytochrome b5 domain, where the rmsf lies between 1.0 and 2.0 Å (Figure 3). Strong fluctuations of up to 3.0 Å were predicted only in the N-terminal region of the cytochrome b5 domain. The Moco and dimerization domains undergo only minor fluctuations of
