CD/MCD/VTVH-MCD Studies of Escherichia coli Bacterioferritin Support a Binuclear Iron Cofactor Site.

Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in Escherichia coli Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O2 oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O2 cycling supports a mechanism in which the binuclear site acts as a cofactor for the O2 reaction, while the mononuclear site binds the substrate Fe(II) that, after its oxidation to Fe(III), migrates to the mineral core.

Iron-nucleated folding of a metalloprotein in high urea: resolution of metal binding and protein folding events.

Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys)(4) site and native protein structure without significant dilution of the chaotrope. This "high-chaotrope" approach was used to examine iron binding and protein folding events using stopped-flow UV-vis absorption and CD spectroscopies. With a 100-fold molar excess of ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe(III)Rd structure was recovered in >90% yield with a t(1/2) of <10 ms. More modest excesses of iron also gave nearly quantitative holoRd formation in 5 M urea but with chronological resolution of iron binding and protein folding events. The results indicate structural recovery in 5 M urea consists of the minimal sequence: (1) binding of ferrous iron to the unfolded apoRd, (2) rapid formation of a near-native ferrous Fe(SCys)(4) site within a protein having no detectable secondary structure, and (3) recovery of the ferrous Fe(SCys)(4) site chiral environment nearly concomitantly with (4) recovery of the native protein secondary structure. The rate of step 2 (and, by inference, step 1) was not saturated even at a 100-fold molar excess of iron. Analogous results obtained for Cys --> Ser iron ligand variants support formation of an unfolded-Fe(SCys)(3) complex between steps 1 and 2, which we propose is the key nucleation event that pulls together distal regions of the protein chain. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys)(4) site from an essentially random coil apoprotein. This high-chaotrope, multispectroscopy approach could clarify folding pathways of other [M(SCys)(3)]- or [M(SCys)(4)]-containing proteins.

Reaction of Desulfovibrio vulgaris two-iron superoxide reductase with superoxide: insights from stopped-flow spectrophotometry.

Stopped-flow mixing of the Desulfovibrio vulgaris two-iron superoxide reductase (2Fe-SOR) containing the ferrous active site with superoxide generates a dead time intermediate whose absorption spectrum is identical to that of a putative ferric-hydroperoxo intermediate previously observed by pulse radiolysis. The dead time intermediate is shown to be a product of reaction with superoxide and to be generated at a much higher proportion of active sites than by pulse radiolysis. This intermediate decays smoothly to the resting ferric active site ( approximately 30 s-1 at 2 degrees C and pH 7) with no other detectable intermediates. Deuterium isotope effects demonstrate that solvent proton donation occurs in the rate-determining step of dead time intermediate decay and that neither of the conserved pocket residues, Glu47 or Lys48, functions as a rate-determining proton donor between pH 6 and pH 8. Fluoride, formate, azide, and phosphate accelerate decay of the dead time intermediate and for azide or fluoride lead directly to ferric-azido or -fluoro complexes of the active site, which inhibit Glu47 ligation. A solvent deuterium isotope effect is observed for the azide-accelerated decay, and the decay rate constants are proportional to the concentrations and pKa values of HX (X- = F-, HCO2-, N3-). These data indicate that the protonated forms of the anions function analogously to solvent as general acids in the rate-determining step. The results support the notion that the ferrous SOR site reacts with superoxide by an inner sphere process, leading directly to the ferric-hydroperoxo intermediate, and demonstrate that the decay of this intermediate is subject to both specific- and general-acid catalysis.