In vitro Reconstitution of the Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

LEONARDO CURATTI; JOSE A. HERNANDEZ; DEHUA ZHAO ; PAUL W. LUDDEN W; LUIS M. RUBIO

Rosario

Conferencia; V Congreso Anual de la Sociedad Argentina de Microbiología General; 2008

Sociedad Argentina de Microbiología General

The biological conversion of atmospheric N2 to NH3 takesplace according to the following reaction: N2 + 8e- + 16MgATP + 2 NH3 + H2 + 16MgADP + 16 Pi and is an essentialstep of the®8 H+ biogeochemical cycle of nitrogen thatsupports life on Earth. The major part of biological nitrogenfixation is catalyzed by the molybdenum nitrogenase enzyme,which is composed of two distinct proteins: dinitrogenase(NifDK) and dinitrogenase reductase (NifH). Dinitrogenasecarries at its active site one of the most complex biologicalmetalloclusters known to date, the iron-molybdenum cofactor(FeMo-co), composed of seven Fe, nine S, one Mo, oneR-homocitrate, and one light unidentified atom.Genetic and biochemical analysis of nitrogen fixation (nif) genesand proteins, mainly in Azotobacter vinelandii and Klebsiellapneumoniae, led to the identification of at least 11 genes (nifUSBQ-ENX-V-H-Y and nafY) proposed to be involved in thebiosynthesis and insertion of FeMo-co into apo-dinitrogenase.We have accomplished the purification of the most criticalproteins for the biosynthesis of FeMo-co, which has provided anunprecedented opportunity to obtain direct biochemicalevidence to support and extend the current working model forFeMo-co biosynthesis and apo-NifDK activation. We show the invitro conversion of apo-NifDK into catalytically competent holo-NifDK in a composition-defined reaction mixture containingpure NifB, NifEN, , NifH, and apo-NifDK as protein factors,along with Na2MoO4, R-homocitrate, (NH4)2Fe(SO4)2, Na2S,SAM, Mg-ATP, and DTH. These results gave strong anddefinitive support to the current model of FeMo-co biosynthesisin which NifB, NifEN and NifH constitute a catalytic corecomprising all the critical steps for the chemical reactions for theassembly of the cofactor from Fe, S, Na2MoO4, R-homocitrate.Nevertheless, non-essential Nif proteins for in vitro synthesis ofFeMoCo play critical roles, especially in vivo, for the assembly ofthe cofactor and may function as the physiological donors of thesubstrates of the pathway. To this regard, we have providedevidence for the participation of NifS and NifU in thebiosynthesis of the iron-sulfur cluster core of FeMo-co (NifB-co)and for NifQ as a specific molybdenum donor