Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes.

Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donor ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.

Unique effect of Cu(II) in the metal-induced amyloid formation of ß-2-microglobulin.

ß-2-Microglobulin (ß2m) forms amyloid fibrils in the joints of patients undergoing hemodialysis treatment as a result of kidney failure. In the presence of stoichiometric amounts of Cu(II), ß2m self-associates into discrete oligomeric species, including dimers, tetramers, and hexamers, before ultimately forming amyloid fibrils that contain no copper. To improve our understanding of whether Cu(II) is unique in its ability to induce ß2m amyloid formation and to delineate the coordinative interactions that allow Cu(II) to exert its effect, we have examined the binding of Ni(II) and Zn(II) to ß2m and the resulting influence that these metals have on ß2m aggregation. We find that, in contrast to Cu(II), Ni(II) does not induce the oligomerization or aggregation of ß2m, while Zn(II) promotes oligomerization but not amyloid fibril formation. Using X-ray absorption spectroscopy and new mass spectrometry-related techniques, we find that different binding modes are responsible for the different effects of Ni(II) and Zn(II). By comparing the binding modes of Cu(II) with Ni(II), we find that Cu(II) binding to Asp59 and the backbone amide between the first two residues of ß2m are important for allowing the formation of amyloid-competent oligomers, as Ni(II) appears not to bind these sites on the protein. The oligomers formed in the presence of Zn(II) are permitted by this metal's ability to bridge two ß2m units via His51. These oligomers, however, are not able to progress to form amyloid fibrils because Zn(II) does not induce the required structural changes near the N-terminus and His31.

Direct evidence of active-site reduction and photodriven catalysis in sensitized hydrogenase assemblies.

We report photocatalytic H(2) production by hydrogenase (H(2)ase)-quantum dot (QD) hybrid assemblies. Quenching of the CdTe exciton emission was observed, consistent with electron transfer from the quantum dot to H(2)ase. GC analysis showed light-driven H(2) production in the presence of a sacrificial electron donor with an efficiency of 4%, which is likely a lower limit for these hybrid systems. FTIR spectroscopy was employed for direct observation of active-site reduction in unprecedented detail for photodriven H(2)ase catalysis with sensitivity toward both H(2)ase and the sacrificial electron donor. Photosensitization with Ru(bpy)(3)(2+) showed distinct FTIR photoreduction properties, generating all of the states along the steady-state catalytic cycle with minimal H(2) production, indicating slow, sequential one-electron reduction steps. Comparing the H(2)ase activity and FTIR results for the two systems showed that QDs bind more efficiently for electron transfer and that the final enzyme state is different for the two sensitizers. The possible origins of these differences and their implications for the enzymatic mechanism are discussed.

Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens.

Previous studies with Geobacter sulfurreducens have demonstrated that OmcS, an abundant c-type cytochrome that is only loosely bound to the outer surface, plays an important role in electron transfer to Fe(III) oxides as well as other extracellular electron acceptors. In order to further investigate the function of OmcS, it was purified from a strain that overproduces the protein. Purified OmcS had a molecular mass of 47015 Da, and six low-spin bis-histidinyl hexacoordinated heme groups. Its midpoint redox potential was -212 mV. A thermal stability analysis showed that the cooperative melting of purified OmcS occurs in the range of 65-82 °C. Far UV circular dichroism spectroscopy indicated that the secondary structure of purified OmcS consists of about 10% α-helix and abundant disordered structures. Dithionite-reduced OmcS was able to transfer electrons to a variety of substrates of environmental importance including insoluble Fe(III) oxide, Mn(IV) oxide and humic substances. Stopped flow analysis revealed that the reaction rate of OmcS oxidation has a hyperbolic dependence on the concentration of the studied substrates. A ten-fold faster reaction rate with anthraquinone-2,6-disulfonate (AQDS) (25.2 s⁻¹) was observed as compared to that with Fe(III) citrate (2.9 s⁻¹). The results, coupled with previous localization and gene deletion studies, suggest that OmcS is well-suited to play an important role in extracellular electron transfer.

Cysteine dioxygenase: structure and mechanism.

Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Crystal structures of mouse, rat, human and bacterial CDO have recently become available and provide significant mechanistic insights. Unlike most non-heme Fe(II) dioxygenases, coordination of the Fe in CDO deviates from the 2-His-1-carboxylate facial triad archetype and instead adopts a His3 facial triad. This change is expected to have an influence on oxygen activation by the catalytic site. The structures also reveal the presence of a cysteinyltyrosine (Tyr157-Cys93) post-translational modification near the active site. Kinetic studies of mutant CDOs reveal that the cysteine residue is less critical than the tyrosine for enzyme activity. Inconsistencies about the details of the active site and the nature of substrate binding exist and are discussed. Herein we review the structural biology along with relevant kinetics studies that have been conducted on CDO for insights into the reaction mechanism of this novel non-heme iron dioxygenase.

Substituent effects on nitrosyl iron corrole complexes Fe(Ar3C)(NO).

A series of nitrosyl tris(5,10,15-aryl)corrolate complexes of iron(III) Fe(Ar3C)(NO) with different substituents on the aryl groups have been prepared, and certain spectroscopic and reaction properties were compared. The cyclic voltammetric analysis of the various Fe(Ar3C)(NO) complexes demonstrated that both the one-electron oxidation and one-electron reduction potentials respond in systematic and nearly identical trends relative to the electron-donor properties of the substituents. A similar pattern was seen in the nitrosyl stretching frequency, nu(NO), which modestly decreased with the stronger donor substituents. Flash photolysis of Fe(Ar3C)(NO) solutions in toluene leads to NO dissociation followed by rapid [NO]-dependent decay of the transients formed (presumably Fe(Ar3C)) to regenerate the original spectra. As was seen in an earlier flash photolysis study of Fe(TNPC)(NO) (TNPC3- = 5,10,15-tris(4-nitro-phenyl)corrolate; Joseph, C.; Ford, P. C. J. Am. Chem. Soc. 2005, 127, 6737-6743), the second-order rate constants, k(NO), are all much faster ((1-9) x 10(8) M(-1) s(-1) at 298 K) than those for analogous iron(III) complexes of porphyrins. However, on a more microscopic level there is no obvious pattern in these rates with respect to the donor properties of the aryl ring substituents. The high reactivity of the ferric triarylcorrolates with NO data is interpreted in terms of the strongly electron-donating character of the Ar3C3- ligand and the quartet electronic configuration of the Fe(Ar3C) intermediate.

The remarkable axial lability of iron(III) corrole complexes.

Flash photolysis of nitrosyl tris(aryl)corrolate complexes of iron(III), Fe(Ar(3)C)(NO) (Ar(3)C(3-) = 5,10,15-tris(4-nitro-phenyl)corrolate (TNPC(3-)), 5,10,15-tris(phenyl)corrolate (TPC(3-)) or 5,10,15-tris(4-tolyl)corrolate (H(3)TTC(3-))) leads to NO labilization. This is followed by the rapid reaction of NO with Fe(III)(C) to regenerate the starting complex. The second-order rate constants for the back reactions (k(NO)) were determined to be many orders of magnitude faster than the corresponding reactions of ferric porphyrin complexes and indeed are reminiscent of the very large values seen for those of the corresponding ferrous porphyrin analogues. These data are interpreted in terms of the strongly electron-donating character of the trianionic corrolate ligand and the likely triplet electronic configuration of the iron(III) complex. These reduce the affinity of the metal centers to Lewis bases to the extent that axial ligands bind very weakly or not at all. This property is illustrated by the nearly identical k(NO) values ( approximately 10(9) M(-1) s(-1) at 295 K) recorded for the back reaction of Fe(III)(TNPC) with NO after flash photolysis of Fe(TNPC)(NO) in toluene solution and in THF solution. Softer Lewis bases have a somewhat greater effect; for example, studies in 1:9 (v:v) acetonitrile:toluene and 1:9 pyridine:toluene gave k(NO) values decreased approximately 33% and approximately 85%, respectively, but these both remain >10(8) M(-1) s(-1). The potential roles of Lewis bases in controlling the dynamics of NO addition to Fe(TNPC) in toluene was investigated in greater detail by determining the rates as a function of pyridine concentration over a wide range (10(-4) to 2.5 M). These data suggest that, while a monopyridine complex, presumably Fe(TNPC)(py), is readily formed (K approximately 10(4) M), this species is about one-sixth as reactive as Fe(TNPC) itself. It appears that a much less reactive bis(pyridine) complex also is formed at high [py] but the equilibrium constant is quite small (<1 M(-1)).