A T14C Variant of Azotobacter vinelandii Ferredoxin I Undergoes Facile [3Fe-4S]0 to [4Fe-4S]2+Conversion in Vitro but Not in Vivo

H Samantha Gao-Sheridan,Mary A Kemper,Reza Khayat,Gareth J Tilley,Fraser A Armstrong,Vandana Sridhar,G Sridhar Prasad,C David Stout,Barbara K Burgess

doi:10.1074/jbc.273.50.33692

Abstract

[4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandii ferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions.

Highlights

One of the most interesting and important features of protein-bound [Fe-S] clusters is their ability to convert from one form to another
For the related iron-responsive element mRNA-binding protein, which is involved in iron homeostasis, the reverse reaction may be the first step on the route to apoprotein production [9]
In a quite different protein, Desulfovibrio gigas ferredoxin II (DgFdII),1 the 3Fe to 4Fe cluster conversion reaction, which is proposed to be controlled by the physiological effector pyruvate, is accompanied by a change in subunit composition of the protein, and the two forms participate in completely different electron transfer pathways [10, 11]

Summary

Introduction

One of the most interesting and important features of protein-bound [Fe-S] clusters is their ability to convert from one form to another (for reviews see Refs. 1–7). As revealed by spectroscopy and x-ray crystallography, when isolated aerobically or anaerobically in the presence of excess dithionite, A. vinelandii ferredoxin I (AvFdI) contains one [4Fe4S]2ϩ/ϩ and one [3Fe-4S]ϩ/0 cluster (29 –31, 38), and the [3Fe4S]ϩ cluster of the protein can be observed in vivo by whole cell EPR (see Ref. 36 and see below).

Results

Conclusion