Combination therapy, or the use of multiple drugs, has been proven to be effective for complex diseases, but the differences in chemical properties and pharmacokinetics can be challenging in term of the loading, delivering, and releasing multiple drugs. Here we demonstrate that we can load and selectively release two different DNA oligonucleotides from two different gold nanorods. DNA was loaded on the nanorods via thiol conjugation. Selective releases were induced by selective melting of gold nanorods via ultrafast laser irradiation at the nanorods' longitudinal surface plasmon resonance peaks. Excitation at one wavelength could selectively melt one type of gold nanorods and selectively release one type

EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of co-substrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a “supernucleophilic” cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g. Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate “base-on” and “base-off” Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of “base-off” Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.

CobA from Salmonella enterica (SeCobA) is a member of the family of ATP:Co(I)rrinoid adenosyltransferase (ACAT) enzymes that participate in the biosynthesis of adenosylcobalamin by catalyzing the transfer of the adenosyl group from an ATP molecule to a reactive Co(I)rrinoid species transiently generated in the enzyme active site. This reaction is thermodynamically challenging, as the reduction potential of the Co(II)rrinoid precursor in solution is far more negative than that of available reducing agents in the cell (e.g., flavodoxin), precluding nonenzymic reduction to the Co(I) oxidation state. However, in the active sites of ACATs, the Co(II)/Co(I) redox potential is increased by >250 mV via the formation of a unique four-coordinate (4c) Co(II)rrinoid species. In the case of the SeCobA ACAT, crystallographic and kinetic studies have revealed that the phenylalanine 91 (F91) and tryptophan 93 (W93) residues are critical for in vivo activity, presumably by blocking access to the lower axial ligand site of the Co(II)rrinoid substrate. To further assess the importance of the F91 and W93 residues with respect to enzymatic function, we have characterized various SeCobA active-site variants using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our data provide unprecedented insight into the mechanism by which SeCobA converts the Co(II)rrinoid substrate to 4c species, with the hydrophobicity, size, and ability to participate in offset π-stacking interactions of key active-site residues all being critical for activity. The structural changes that occur upon Co(II)rrinoid binding also appear to be crucial for properly orienting the transiently generated Co(I) “supernucleophile” for rapid reaction with cosubstrate ATP.

Nitrosylcobalamin (NOCbl) is readily formed when Co(II)balamin reacts with nitric oxide (NO) gas. NOCbl has been implicated in the inhibition of various B12-dependent enzymes, as well as in the modulation of blood pressure and of the immunological response. Previous studies revealed that among the known biologically relevant cobalamin species, NOCbl possesses the longest bond between the Co ion and the axially bound 5,6-dimethylbenzimidazole base, which was postulated to result from a strong trans influence exerted by the NO ligand. In this study, various spectroscopic (electronic absorption, circular dichroism, magnetic circular dichroism, and resonance Raman) and computational (density functional theory (DFT) and time-dependent DFT) techniques were used to generate experimentally validated electronic structure descriptions for the “base-on” and “base-off” forms of NOCbl. Further insights into the principal Co–ligand bonding interactions were obtained by carrying out natural bond orbital analyses. Collectively, our results indicate that the formally unoccupied Co 3dz2 orbital engages in a highly covalent bonding interaction with the filled NO π* orbital and that the Co–NO bond is strengthened further by sizable π-backbonding interactions that are not present in any other Co(III)Cbl characterized to date. Because of the substantial NO– to Co(III) charge donation, NOCbl is best described as a hybrid of Co(III)–NO– and Co(II)–NO• resonance structures. In contrast, our analogous computational characterization of a related species, superoxocobalamin, reveals that in this case a Co(III)–O2– description is adequate due to the larger oxidizing power of O2 versus NO. The implications of our results with respect to the unusual structural features and thermochromism of NOCbl and the proposed inhibition mechanisms of B12-dependent enzymes by NOCbl are discussed.

The EutT enzyme from Salmonella enterica, a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes, requires a divalent transition metal ion for catalysis, with Fe(II) yielding the highest activity. EutT contains a unique cysteine-rich HX11CCX2C(83) motif (where H and the last C occupy the 67th and 83rd positions, respectively, in the amino acid sequence) not found in other ACATs and employs an unprecedented mechanism for the formation of adenosylcobalamin (AdoCbl). Recent kinetic and spectroscopic studies of this enzyme revealed that residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate (4c) cob(II)alamin [Co(II)Cbl] intermediate in the catalytic cycle. However, it remained unknown which, if any, of the residues in the HX11CCX2C(83) motif bind the divalent metal ion. To address this issue, we have characterized Co(II)-substituted wild-type EutT (EutTWT/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutTWT/Co relative to the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of 4c Co(II)Cbl. Our MCD data of EutTWT/Co also reveal that the Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Additional spectroscopic studies of EutT/Co variants possessing a single alanine substitution at either the His67, His75, Cys79, Cys80, or Cys83 position indicate that Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site.