Cloning and mutational analysis of the gamma gene from Azotobacter vinelandii defines a new family of proteins capable of metallocluster binding and protein stabilization

• Published in: The Journal of biological chemistry 2002-04, Vol.277 (16), p.14299-14305
• Main Authors: Rubio, Luis M, Rangaraj, Priya, Homer, Mary J, Roberts, Gary P, Ludden, Paul W
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Azotobacter vinelandii; Azotobacter vinelandii - genetics; Azotobacter vinelandii - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cloning, Molecular; Dinitrogenase; DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Metalloproteins - genetics; Metalloproteins - metabolism; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; nafY gene; nif gene; NifX protein; NifY protein; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Sequence Analysis, DNA; Time Factors; VnfX protein
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M107289200

Dinitrogenase is a heterotetrameric (alpha(2)beta(2)) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition alpha(2)beta(2)gamma(2), which can be activated in vitro by the addition of FeMo-co. The gamma protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the gamma subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now gamma. In vitro FeMo-co insertion experiments presented in this work demonstrate that gamma stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.