T14/D15 Azotobacter vinelandii Ferredoxin I: Creat
详细信息 查看全文
- 作者：Mary A. Kemper ; H. Samantha Gao-Sheridan ; Binghui Shen ; Jillian L. C. Duff ; Gareth J. Tilley ; Fraser A. Armstrong ; and Barbara K. Burgess
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：September 15, 1998
- 年：1998
- 卷：37
- 期：37
- 页码：12829 - 12837
- 全文大小：213K
- 年卷期：v.37,no.37(September 15, 1998)
- ISSN：1520-4995
文摘
In clostridial-type ferredoxins, each of the two [4Fe-4S]2+/+ clusters receives three of its fourligands from a CysXXCysXXCys motif. Azotobacter vinelandii ferredoxin I (AvFdI) is a seven-ironferredoxin that contains one [4Fe-4S]2+/+ cluster and one [3Fe-4S]+/0 cluster. During the evolution ofthe 7Fe azotobacter-type ferredoxins from the 8Fe clostridial-type ferredoxins, one of the two motifspresent changed to a CysXXCysXXXXCys motif, resulting in the inability to form a 4Fe cluster and theappearance of a 3Fe cluster in that position. In a previous study, we were unsuccessful in using structureas a guide in designing a 4Fe cluster in the 3Fe cluster position of AvFdI. In this study, we have reversedpart of the evolutionary process by deleting two residues between the second and third cysteines. UV/Vis, CD, and EPR spectroscopies and direct electrochemical studies of the purified protein reveal thatthis 
T14/D15 FdI variant is an 8Fe protein containing two [4Fe-4S]2+/+ clusters with reduction potentialsof -466 and -612 mV versus SHE. Whole-cell EPR shows that the protein is present as an 8Fe proteinin vivo. These data strongly suggest that it is the sequence motif rather than the exact sequence or thestructure that is critical for the assembly of a 4Fe cluster in that region of the protein. The new oxygen-sensitive 4Fe cluster was converted in partial yield to a 3Fe cluster. In known ferredoxins and enzymesthat contain reversibly interconvertible [4Fe-4S]2+/+ and [3Fe-4S]+/0 clusters, the 3Fe form always has areduction potential ca. 200 mV more positive than the 4Fe cluster in the same position. In contrast, forT14/D15 FdI, the 3Fe and 4Fe clusters in the same location have extremely similar reduction potentials.
T14/D15 FdI variant is an 8Fe protein containing two [4Fe-4S]2+/+ clusters with reduction potentialsof -466 and -612 mV versus SHE. Whole-cell EPR shows that the protein is present as an 8Fe proteinin vivo. These data strongly suggest that it is the sequence motif rather than the exact sequence or thestructure that is critical for the assembly of a 4Fe cluster in that region of the protein. The new oxygen-sensitive 4Fe cluster was converted in partial yield to a 3Fe cluster. In known ferredoxins and enzymesthat contain reversibly interconvertible [4Fe-4S]2+/+ and [3Fe-4S]+/0 clusters, the 3Fe form always has areduction potential ca. 200 mV more positive than the 4Fe cluster in the same position. In contrast, forT14/D15 FdI, the 3Fe and 4Fe clusters in the same location have extremely similar reduction potentials.