Trace cobalt speciation in bacteria and at enzymic active sites using emission Mössbauer spectroscopy.

57Co emission Mössbauer spectroscopy (EMS) allows the chemical state of cobalt, as influenced by its coordination environment, to be monitored in biological samples at its physiological (trace) concentrations. To draw attention to EMS as a valuable tool for speciation of cobalt in biocomplexes, the process of cobalt(II) metabolism in cells of the plant growth-promoting rhizobacterium Azospirillum brasilense Sp245 was investigated using EMS of 57CoII-doped bacterial cells. EMS measurements also showed 57CoII-activated glutamine synthetase (GS, a key enzyme of nitrogen metabolism, isolated from this bacterium) to have two different cobalt(II) forms at its active sites, in agreement with data available on other bacterial GSs. Chemical after-effects following electron capture by the nucleus of the parent 57CoII during the 57Co-->57Fe transition, which contribute to the formation of a stabilised daughter 57FeIII component along with the nucleogenic 57FeII forms, are also briefly considered.

Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense.

Fully unadenylylated glutamine synthetase (GS) from the endophytic bacterium Azospirillum brasilense Sp245 was isolated and purified. The enzyme was electrophoretically homogeneous and contained strongly bound metal ions, which could not be removed by dialysis. Mn2+, Mg2+, and Co2+ were found to be effective in supporting biosynthetic activity of the A. brasilense GS. Some kinetic properties of Mn2+-activated and Mg2+-activated unadenylylated GS were characterized. Circular dichroism analysis of the enzyme showed that the A. brasilense GS is a highly structured protein: 59% of its residues form alpha-helices and 13% beta-strands. Removal of the metal ions from the A. brasilense GS by treatment with EDTA resulted in alterations in the enzyme secondary structure.