Date of Award:

5-2000

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Chemistry and Biochemistry

Department name when degree awarded

Biochemistry

Committee Chair(s)

Lance C. Seefeldt (Committee Chair)

Committee

Lance C. Seefeldt

Committee

John Peter

Committee

Scott Ensign

Abstract

Nitrogenase is the enzyme that catalyzes the reduction of nitrogen to ammonia in a reaction requiring MgATP hydrolysis. Two component proteins of nitrogenase are the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein).

Nitrogenase contains two nucleotide binding sites. During catalysis, the Fe protein binds two MgATP first. The conformational changes induced upon MgATP binding allow the Fe protein to associate with the MoFe protein. After the formation of the Fe protein-MoFe protein complex, a single electron is transferred from the Fe protein to the MoFe protein, an event that is coupled to MgATP hydrolysis in the Fe protein. The wild-type Fe protein and all the altered Fe proteins studied so far are homodimeric. In order to assess the contribution of each nucleotide binding site in the Fe protein to the events occurring during nitrogenase catalysis, a heterodimeric Fe protein was constructed that has Asp 39 substituted by Asn in one subunit and the other subunit the same as the wild-type Fe protein. Characterization of this heterodimeric Fe protein showed that alterations in the properties of the [4Fe-4S] cluster that occur upon nucleotide binding to the Fe protein are due to the additive effect of each nucleotide binding to the Fe protein. The rates of MgATP hydrolysis and MgATP-dependent primary electron transfer of this heterodimeric Fe protein are intermediate between those of the homodimeric wild-type Fe protein and D39N Fe protein. These observations suggested that each ATP binding site contributes to the rate acceleration of primary electron transfer. After electron transfer, this heterodimeric Fe protein forms a tight complex with the MoFe protein, demonstrating that alteration in one subunit is enough for the formation of a tight nitrogenase complex. When this heterodimeric Fe protein was combined with the MoFe protein, no substrate reduction was detected. Therefore, two functional subunits of the Fe protein are necessary for reduction of substrates.

The mechanism of ATP hydrolysis in the Fe protein was also investigated. Using site-directed mutagenesis, the role of lysine 10 of the Azotobacter vinelandii nitrogenase Fe protein in MgATP hydrolysis was examined. Changing Lys 10 of the protein to Arg resulted in an Fe protein that hydrolyzed MgATP at a rate 3% that of the wild-type Fe protein. The affinities of the K10R Fe protein for nucleotides and the changes in the properties of the [4Fe-4S] cluster of the K10R Fe protein upon nucleotide binding were compared with those of the wild-type Fe protein. These results indicated that in the absence of the MoFe protein, the interactions of the K10R Fe protein with nucleotides are similar to the wild-type Fe protein. After the Fe protein-MoFe protein complex formation, the dramatic decrease in the rate of MgATP hydrolysis of the K10R Fe protein indicated a role of Lys 10 in ATP hydrolysis. This conclusion is consistent with the X-ray crystal structure of the nitrogenase complex stabilized by the AlF4-•ADP, where Lys 10 is proposed to facilitate product formation in ATP hydrolysis.

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