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Specific interaction of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with the nitrogenase MoFe protein.

Abstract：

:Putative physiological partners of the [2Fe-2S] ferredoxin from Clostridium pasteurianum have been searched by running crude soluble extracts of this bacterium through an affinity column to which the [2Fe-2S] ferredoxin had been covalently bound. Subsequent washing of the column with buffers of increasing ionic strength revealed a strong and specific interaction of the ferredoxin with the MoFe protein of nitrogenase. This interaction was further investigated by performing cross-linking experiments with mixtures of the two purified proteins in solution. Analysis of the reactions by SDS-polyacrylamide gel electrophoresis evidenced only two covalently linked products. These were identified by N-terminal sequencing as the alpha and beta subunits of the MoFe protein, each cross-linked to a single polypeptide chain of the ferredoxin. This result, taking into account the dimeric structure of the ferredoxin in solution, strongly suggests an interaction of the ferredoxin with the MoFe protein at a site contributed to by both subunits of the MoFe protein. The ionic strength dependence of the interaction evidenced by affinity chromatography was confirmed in the cross-linking reactions, and its specificity was assessed by showing that no cross-linking occurred when the [2Fe-2S] C. pasteurianum ferredoxin was denatured or replaced by spinach ferredoxin or by clostridial rubredoxin, or when the MoFe protein from C. pasteurianum was either inactivated or replaced by its counterpart from Azotobacter vinelandii. It has also been observed that the ferredoxin inhibits cross-linking between the nitrogenase Fe protein and the MoFe protein, which suggests overlapping binding sites of the ferredoxin and of the Fe protein on the MoFe protein. Cross-linking experiments implementing a number of molecular variants of the [2Fe-2S] C. pasteurianum ferredoxin demonstrated that glutamate residues 31, 34, and 38 are important contributors to the interaction with the MoFe protein.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Golinelli MP,Gagnon J,Meyer J

doi

10.1021/bi970528p

subject

Has Abstract

pub_date

1997-09-30 00:00:00

pages

11797-803

issue

eissn

0006-2960

issn

1520-4995

pii

bi970528p

journal_volume

pub_type

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Specific interaction of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with the nitrogenase MoFe protein.