Ni(2+) transport and accumulation in Rhodospirillum rubrum.
The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO.
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
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RK
Watt
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Ludden
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002248:Carbon Monoxide / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D019811:Hydroxylamine / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D009532:Nickel / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D012247:Rhodospirillum rubrum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019810:Sodium Azide / Q000494:pharmacology
D002413:Cations, Divalent; D008670:Metals; D009097:Multienzyme Complexes; D019811:Hydroxylamine; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D015759:Ionomycin; D009532:Nickel; D002248:Carbon Monoxide; D019810:Sodium Azide; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D006180:Proton-Translocating ATPases; D019297:2,4-Dinitrophenol