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.

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.

We report photo-catalytic H2 production by hydrogenase (H2ase)-quantum dot (QD) hybrid assemblies. Quenching of the CdTe exciton emission is observed, consistent with electron transfer from quantum dot to H2ase. GC analysis shows light driven H2 production in the presence of a sacrificial electron donor with an efficiency of 4%, which is likely a lower limit to these hybrid systems. FTIR was employed for direct observation of active site reduction in unprecedented detail for photo-driven H2ase catalysis with sensitivity towards both H2ase and sacrificial electron donor. Photosensitization with Ru(bpy)32+ shows distinct FTIR photo- reduction properties generating all states along the steady-state catalytic cycle with minimal H2 production indicating slow, sequential one electron reduction steps. Comparing H2ase activity and FTIR results of both systems shows that QDs bind more efficiently for electron transfer and 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.

β-2-Microglobulin (β2m) 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), β2m 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 β2m 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 β2m and the resulting influence that these metals have on β2m aggregation. We find that, in contrast to Cu(II), Ni(II) does not induce the oligomerization or aggregation of β2m, 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 β2m 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 β2m 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.

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)).