The role of the MoFe protein alpha-125Phe and beta-125Phe residues in Azotobacter vinelandii MoFe protein-Fe protein interaction.
Site-directed mutagenesis and gene-replacement techniques were used to substitute alanine for the MoFe protein alpha- and beta-subunit phenylalanine-125 residues both separately and in combination. These residues are located on the surface of the MoFe protein near the pseudosymmetric axis of symmetry between the alpha- and beta-subunits. Altered MoFe proteins that contain an alanine substitution at only one of the respective positions exhibit proton reduction activities of about 25-50% when compared to that of the wild-type protein. The lower level of proton reduction also corresponds with decreases in the rates of MgATP hydrolysis. The MoFe protein which contains alanine substitutions in both the alpha- and beta- subunits did not exhibit any proton reduction activity or MgATP hydrolysis. Stopped flow spectrophotometry of the singly substituted MoFe proteins indicate primary electron transfer rate constants approximately an order of magnitude slower than what is observed for wild-type MoFe protein, while no primary electron transfer is observed for the doubly substituted MoFe protein. The doubly substituted MoFe protein is able to interact with the Fe protein as shown by chemical crosslinking experiments. However, this protein does not form a tight complex with the Fe protein when treated with MgADP-AlF4- or when using the altered 127delta Fe protein. Stopped flow spectrophotometry was also used to quantitate the first-order dissociation rate constants for the two component proteins. These results suggest that the 125Phe residues are involved in an early event(s) that occurs upon component protein docking and could be involved in eliciting MgATP hydrolysis.
Christiansen
J
J
Department of Biochemistry, Fralin Biotechnology Center, Virginia Tech, Blacksburg 24061, USA.
Chan
J M
JM
Seefeldt
L C
LC
Dean
D R
DR
D000255:Adenosine Triphosphate / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009586:Nitrogen Fixation / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*
D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C029130:nitrogenase reductase