Internal dynamics of the 3-Pyrroline-N-Oxide ring in spin-labeled proteins

Site-directed spin labeling is a versatile tool to study structure as well as dynamics of proteins using EPR spectroscopy. Methanethiosulfonate (MTS) spin labels tethered through a disulfide linkage to an engineered cysteine residue were used in a large number of studies to extract structural as well as dynamic information on the protein from the rotational dynamics of the nitroxide moiety. The ring itself was always considered to be a rigid body. In this contribution, we present a combination of high-resolution X-ray crystallography and EPR spectroscopy of spin-labeled protein single crystals demonstrating that the nitroxide ring inverts fast at ambient temperature while exhibiting nonplanar conformations at low temperature. We have used quantum chemical calculations to explore the potential energy that determines the ring dynamics as well as the impact of the geometry on the magnetic parameters probed by EPR spectroscopy

Functional Studies on <i>Oligotropha carboxidovorans</i> Molybdenum–Copper CO Dehydrogenase Produced in <i>Escherichia coli</i>

The Mo/Cu-dependent CO dehydrogenase (CODH) from <i>Oligotropha carboxidovorans</i> is an enzyme that is able to catalyze both the oxidation of CO to CO₂ and the oxidation of H₂ to protons and electrons. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of <i>O. carboxidovorans</i> CODH in <i>Escherichia coli</i>. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from <i>O. carboxidovorans</i>. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H₂ oxidation of Mo/Cu-dependent CODHs in the future

Active Site of the NAD⁺‑Reducing Hydrogenase from <i>Ralstonia eutropha</i> Studied by EPR Spectroscopy

Pulsed ENDOR and HYSCORE measurements were carried out to characterize the active site of the oxygen-tolerant NAD⁺-reducing hydrogenase of <i>Ralstonia eutropha</i>. The catalytically active Ni_a-C state exhibits a bridging hydride between iron and nickel in the active site, which is photodissociated upon illumination. Its hyperfine coupling is comparable to that of standard hydrogenases. In addition, a histidine residue could be identified, which shows hyperfine and nuclear quadrupole parameters in significant variance from comparable histidine residues that are conserved in standard [NiFe] hydrogenases, and might be related to the O₂ tolerance of the enzyme

Does Tyrosine Protect <i>S. coelicolor</i> Laccase from Oxidative Degradation or Act as an Extended Catalytic Site?

We have investigated the roles of tyrosine (Tyr) and tryptophan (Trp) residues in the four-electron reduction of oxygen catalyzed by Streptomyces coelicolor laccase (SLAC). During normal enzymatic turnover in laccases, reducing equivalents are delivered to a type 1 Cu center (CuT1) and then are transferred over 13 Å to a trinuclear Cu site (TNC: (CuT3)2CuT2) where O2 reduction occurs. The TNC in SLAC is surrounded by a large cluster of Tyr and Trp residues that can provide reducing equivalents when the normal flow of electrons is disrupted. Prior studies by Canters and co-workers [J.Am.Chem.Soc.2009, 131 (33), 11680-11682] have shown that when O2 reacts with a reduced SLAC variant lacking the CuT1 center, a Tyr108• radical near the TNC forms rapidly. We have found that the Tyr108• radical is reduced 10 times faster than CuT12+ by excess ascorbate, possibly because of radical transfer along Tyr/Trp chains

Steady-state kinetic parameters of mAOX3 wild type and mAOX3 variants containing the FeS-domains of mAOX1.

<p>µM DCPIP was used as terminal electron receptor.¹ 100 </p><p>² Molecular oxygen in air saturated buffer was used as terminal electron acceptor.</p><p>°C; k_cat was corrected for molybdenum content³ Determined in 50 mM Tris HCl pH 8.00 at 37</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082285#pone.0082285-Coelho1" target="_blank">[23]</a> and corrected for molybdenum content.⁴ Values from </p

Determination of the cofactor composition of the mAOX3 variants.

<p>µM molybdenum/µM enzyme) and iron (µM 2 x [2Fe2S]/µM enzyme) contents were determined by ICP-OES (see Experimental procedures).¹ Molybdenum (</p><p>%.² Determined after conversion to the stable oxidized fluorescent product FormA. mAOX3-WT was set to 100</p><p> Relative molybdopterin (MPT) saturation, molybdenum and iron saturation and ratio of absorbance at 280 nm, 444 nm and 550 nm of mAOX3 variants in comparison to reported values for the wild type enzyme.</p

Interactions established by MD simulation within the metallic active center in the wild type and variant enzymes.

<p>*only available in the respective mutated enzymes.</p><p>a) substrate is hypoxanthine</p><p>b) substrate is benzaldehyde</p

Steady-state kinetic parameters for mAOX3 wild type and mAOX3 variants containing numerous amino acids present in the active site of bovine XOR.

<p>µM DCPIP in 50 mM Tris (HCl) pH 8.00 at 37°C; k_cat was corrected for molybdenum content¹ Determined in the presence of 100 </p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082285#pone.0082285-Coelho1" target="_blank">[23]</a> and corrected for molybdenum content.² Values from </p><p> n.d. none detectable.</p><p>⁴ phthalazine:DCPIP reaction was biphasic.</p

Active site structure of mAOX3.

<p>A, Stick representation of E1266 and residues exchanged in mAOX3-“active site1”-K889H in the crystal structure of mAOX3 WT (pdb:3ZYV). B, Stick representation of residues of desulfurated bXO (pdb:3EUB) corresponding to the amino acids in Panel A. C, Stick representation of residues in mAOX3 corresponding to the residues shown in Panel D. Y885 builds hydrogen-bonds to the backbone of G1013 and K889, indicated by yellow dotted lines D, Stick representation of residues involved in a hydrogen network at the entrance to the active site of bXOR. The hydrogen-bonding network is established by R880, G1006, I1007, S1008 and N1015 in bXOR represented by yellow dotted lines. Through the interactions, the position and orientation of T1010 is altered in comparison to K1016 of mAOX3 in panel C. Figures were created using MacPymol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082285#pone.0082285-Schrodinger1" target="_blank">[56]</a>Ver. 0.99rc6.</p

Steady-state kinetic parameters of mAOX3 wildtype and mAOX3 variants containing single amino acid exchanges in the active site.

<p>µM DCPIP was used as terminal electron receptor.¹ 100 </p><p>² Molecular oxygen in air saturated buffer was used as terminal electron acceptor.</p><p>°C; k_cat was corrected for molybdenum content³ Determined in 50 mM Tris (HCl) pH 8.00 at 37</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082285#pone.0082285-Coelho1" target="_blank">[23]</a> and corrected for molybdenum content.⁴ Values from </p><p> n.d.: non detectable</p