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J. Biol. Chem., Vol. 277, Issue 16, 14299-14305, April 19, 2002
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From the Departments of Dinitrogenase is a heterotetrameric
( The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF302049.
Cloning and Mutational Analysis of the
Gene from
Azotobacter vinelandii Defines a New Family of Proteins
Capable of Metallocluster Binding and Protein Stabilization*
,,
Biochemistry and
§ Bacteriology and the Center for the Study of Nitrogen
Fixation, College of Agricultural and Life Sciences, University of
Wisconsin-Madison, Madison, Wisconsin 53706
2
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 22
2, which can
be activated in vitro by the addition of FeMo-co. The 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 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 . In vitro FeMo-co insertion
experiments presented in this work demonstrate that 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.
*
This work was supported by Grant 35332 from the NIGMS,
National Institutes of Health (to P. W. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry, 433 Babcock Dr., University of Wisconsin-Madison,
Madison, WI 53706. Tel.: 608-262-6859; Fax: 608-262-3453;
E-mail: pludden@cals.wisc.edu.
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