PubMed · 10543113
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S Nomoto. 1999. [Cobalt (Co)].. https://pubmed.ncbi.nlm.nih.gov/10543113/
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Cobalt influences the methanol metabolism of Desulfofundulus kuznetsovii TPOSR, specifically by modulating the activity of one of its alcohol dehydrogenases (ADH), Adh1. However, the effects of cobalt on the broader proteome of strain TPOSR, as well as the utilization of alcohols besides methanol, remain unexplored. Here, proteomic analyses of strain TPOSR grown with and without cobalt on different alcohol substrates show that cobalt starvation impacts multiple cellular processes, including cobalamin biosynthesis, iron-sulphur cluster assembly and, most prominently, energy metabolism as indicated by altered abundances of hydrogenases and NAD(P)-dependent oxidoreductases. Despite the presence of six ADH-encoding genes in the genome, Adh1 is the dominant ADH during growth not only on methanol but also on several primary alcohols and diols (ethanol, 1-propanol, 1,2-propanediol, 1,3-propanediol, butanol, pentanol and heptanol). Enzymatic assays with purified Adh1 confirm activity with these substrates, except 1,3-propanediol, and show no activity toward secondary alcohols (2-propanol and 2-butanol). Comparative proteomics analyses of other sulphate-reducing microorganisms (SRMs), namely Desulfofundulus australicum and Solidesulfovibrio carbinolicus, further indicate that methanol and ethanol oxidation in SRMs is mediated by a single ADH/AOR pair. Together, these findings highlight the central role of cobalt in alcohol metabolism in strain TPOSR and identify conserved ADH/AOR enzymes as promising candidates for biotechnological applications.
Three hexadentate, asymmetric pendent arm macrocycles containing a 1,4,7-triazacyclononane-1,4-diacetate backbone and a third, N-bound phenolate or thiophenolate arm have been synthesized. In [L(1)](3)(-) the third arm is 3,5-di-tert-butyl-2-hydroxybenzyl, in [L(2)](3)(-) it is 2-mercaptobenzyl, and in [L(3)](3)(-) it is 3,5-di-tert-butyl-2-mercaptobenzyl. With trivalent metal ions these ligands form very stable neutral mononuclear complexes [M(III)L(1)] (M = Ga, Fe, Co), [M(III)L(2)] (M = Ga, Fe, Co), and [M(III)L(3)] (M = Ga, Co) where the gallium and cobalt complexes possess an S = 0 and the iron complexes an S = (5)/(2) ground state. Complexes [CoL(1)].CH(3)OH.1.5H(2)O, [CoL(3)].1.17H(2)O, [FeL(1)].H(2)O, and [FeL(2)] have been characterized by X-ray crystallography. Cyclic voltammetry shows that all three [M(III)L(1)] complexes undergo a reversible, ligand-based, one-electron oxidation generating the monocations [M(III)L(1)(*)](+) which contain a coordinated phenoxyl radical as was unambiguously established by their electronic absorption, EPR, and Mössbauer spectra. In contrast, [M(III)L(2)] complexes in CH(3)CN solution undergo an irreversible one-electron oxidation where the putative thiyl radical monocationic intermediates dimerize with S-S bond formation yielding dinuclear disulfide species [M(III)L(2)-L(2)M(III)](2+). [GaL(3)] behaves similarly despite the steric bulk of two tertiary butyl groups at the 3,5-positions of the thiophenolate, but [Co(III)L(3)] in CH(2)Cl(2) at -20 to -61 degrees C displays a reversible one-electron oxidation yielding a relatively stable monocation [Co(III)L(3)(*)](+). Its electronic spectrum displays intense transitions in the visible at 509 nm (epsilon = 2.6 x 10(3) M(-)(1) cm(-)(1)) and 670sh, 784 (1.03 x 10(3)) typical of a phenylthiyl radical. The EPR spectrum of this species at 90 K proves the thiyl radical to be coordinated to a diamagnetic cobalt(III) ion (g(iso) = 2.0226; A(iso)((59)Co) = 10.7 G).
Glutathionylcobalamin (gamma-glutamylcysteinylglycinylcobalamin; gamma-GluCysGly-Cbl) is a natural product which functions as an intermediate in the biosynthesis of the active B(12) coenzymes adenosylcobalamin and methylcobalamin. Of interest to the present studies is glutathionylcobalamin's unique stability in comparison to other thiolatocobalamins, notably the > or =6 x 10(4) fold less stable cysteinylcobalamin, Cys-Cbl. In order to determine which parts of the glutathione tripeptide contribute to the overall stability of glutathionylcobalamin, two cysteine-containing dipeptides, which are truncated versions of glutathione, were used to synthesize their corresponding cobalamins, specifically gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl). As with glutathionylCbl, the dipeptide gamma-GluCys-Cbl forms a stable thiolatocobalamin. However and most interestingly, CysGly-Cbl is observed to be unstable much like Cys-Cbl. The results require that the extra stability of glutathionylcobalamin and its congeners, compared to cysteinylcobalamin and its analogues, must be derived from destabilization by the gamma-NH(3)(+) group in cysteinylcobalamin, or stabilization by the gamma-NHC(=O)- amide linkage in glutathionylcobalamin, or both. To probe any ground-state structural basis for the possible stabilization in gamma-GluCys-containing cobalamins, gamma-GluCys-Cbl was crystallized and yielded the first X-ray structural determination of a true thiolatocobalamin, and only the second structure of a cobalamin containing a Co-S bond, the first example being Randaccio and co-workers' 1999 structure of the thioketone complex, thioureacobalamin, (NH(2))(2)CSCbl. Key features of the structure of gamma-glutamylcysteinylcobalamin include (i) a normal Co-S bond length of 2.267(2) A, (ii) a Co-N(axial) bond length of 2.049(6) A, (iii) two alternate conformations of the gamma-glutamylcysteinyl moiety, and (iv) folding of the corrin ring upward by 24.2 degrees, the highest degree of folding yet observed for a cobalamin. These results do not show any strong stabilization (e.g., no shortened Co-S bond), although it is not clear for certain what the effect is (stabilizing or destabilizing) of the elongated Co-N(axial) bond; instead, the crystallographic results suggest that the metastable Cys-Cbl probably has a Co-S cleavage transition state that is stabilized (along with, possibly, any ground-state destabilization of the Co-S bond). Overall, the results strongly suggest that placing a positive charge on the gamma-NH(3)(+) stabilizes the Co-S bond cleavage transition state, thereby setting the stage for the needed full thermolysis product and kinetic studies-as a function of the axial-base on-off equilibrium-that will be required to understand in even greater detail the unique stability of glutathionyl- (gamma-glutamylcysteinylglycinyl-) and gamma-glutamylcysteinylcobalamins.