Complex Formation with the Activator RACo Affects the Corrinoid Structure of CoFeSP

Activation of the corrinoid [Fe-S] protein (CoFeSP), involved in reductive CO₂ conversion, requires the reduction of the Co­(II) center by the [Fe-S] protein RACo, which according to the reduction potentials of the two proteins would correspond to an uphill electron transfer. In our resonance Raman spectroscopic work, we demonstrate that, as a conformational gate for the corrinoid reduction, complex formation of Co­(II)­FeSP and RACo specifically alters the structure of the corrinoid cofactor by modifying the interactions of the Co­(II) center with the axial ligand. On the basis of various deletion mutants, the potential interaction domains on the partner proteins can be predicted

EPR linewidths and g-values of FeSI and FeSII from mAOX1.

a<p>AOX wild-type from rabbit liver, values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005348#pone.0005348-Stesmans1" target="_blank">[36]</a>.</p>b<p>g-strain was included in the simulation with 0.01 for g_z .</p>c<p>g-strain was included in the simulation with 0.04 for g_x . Estimated error of g-values: ±0.004 for FeSI and ±0.008 for FeSII.</p

Steady-state kinetic parameters of recombinant mAOX1 and variants with different aldehyde and purine substrates.

a<p>Apparent kinetic parameters were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5 by varying the concentration of substrate in the presence of 100 µM DCPIP as electron acceptor.</p><p>n.d., none was detectable.</p><p>-, not determined.</p

Steady-state kinetic parameters of recombinant <i>R. capsulatus</i> XDH and variants with different aldehyde and purine substrates.

a<p>Apparent kinetic parameters were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5 by varying the concentration of substrate in the presence of 100 µM DCPIP as electron acceptor.</p><p>n.d., none was detectable.</p><p>-, not determined.</p

EPR spectra of mAOX1 wild-type.

<p>Experimental cw-EPR spectra of dithionite-reduced mAOX1 wild-type samples at pH 7.0 (trace a) together with the corresponding simulation (trace b). For simulation parameters see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005348#pone-0005348-t003" target="_blank">Table 3</a>. The flavin semiquinone was simulated with an isotropic g-value of g_iso = 2.0 and 1.9 mT (trace e). (MoV) was neglected in all simulations. (a) mAOX1 wild-type; (b) simulation of complete spectrum; (c) simulation of FeSI; (d) simulation of FeSII; (e) simulation of FAD. Experimental conditions: T = 20 K, 1 mW microwave power, 1 mT modulation amplitude, 12.5 kHz modulation frequency.</p

Characterization of wild-type mAOX1 by UV-VIS absorption spectroscopy.

<p>Spectra of 7 µM of the air-oxidized mAOX1 in 50 mM Tris, 1 mM EDTA, pH 7.5, under anaerobic conditions.</p

Purification of recombinant mAOX1 after expression in <i>E. coli</i> TP1000 cells.

a<p>Total protein was quantified with the Bradford assay.</p>b<p>The activity was measured by monitoring the decrease in absorption at 600 nm in the presence of 500 µM benzaldehyde and 100 µM DCPIP.</p>c<p>Specific enzyme activity (units/mg) is defined as the oxidation of 1 µM benzaldehyde per min and mg of enzyme under the assay conditions.</p

Native PAGE of mAOX1 wild-type and variants after purification.

<p>Purified enzymes were analyzed by 7% native PAGE. Each lane contained 6 µg of purified enzyme: lane 1, mAOX1 wild-type; lane 2, mAOX1-V806E; lane 3, mAOX1-M884R; lane 4, mAOX1-V806E/M884R; lane 5, mAOX1-E1265Q.</p

Determination of the Moco and iron content of mAOX1 and <i>R. capsulatus</i> XDH and variants.

a<p>Moco was quanitfied as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005348#s4" target="_blank">Materials and Methods</a>. Moco content of wild-type mAOX1 and <i>R. capsulatus</i> XDH was set to the calculated molybdenum content determined by ICP-OES, and Moco determined as Form A in the AOX1 variants was compared to that value.</p>b<p>Iron was determined by ICP-OES as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005348#s4" target="_blank">Materials and Methods</a>.</p

Purification of mAOX1 after heterologous expression in <i>E. coli</i>. 12% SDS-PAGE analysis of purification of mAOX1.

<p>The protein was purified after Ni-NTA, Superose 12 and benzamidine sepharose 6B. Purified mAOX1 displays a molecular mass of 150 kDa on SDS-PAGE, the three bands with sizes of 120 kDa, 80 kDa, and 50 kDa correspond to degradation products of mAOX1, as determined by mass spectrometry.</p