Human Ferredoxin: Overproduction in Escherichia coli, Reconstitution in Vitro, and Spectroscopic Studies of Iron-Sulfur Cluster Ligand Cysteine-to-Serine Mutants † Bin Xia, ‡,§ Hong Cheng, §,| Vahe Bandarian, § George H. Reed, ‡,§,⊥ and John L. Markley* ,‡,§ Graduate Program in Biophysics, Department of Biochemistry, and Institute for Enzyme Research, UniVersity of WisconsinsMadison, Madison, Wisconsin 53706 ReceiVed February 26, 1996; ReVised Manuscript ReceiVed May 16, 1996 X ABSTRACT: Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small iron-sulfur protein with one [2Fe-2S] cluster. It functions, as do other vertebrate ferredoxins, to transfer electrons during the processes of steroid hormone synthesis. A DNA fragment encoding the mature form of human ferredoxin was cloned into an expression vector under control of the T7 RNA polymerase/promoter system. The protein was overproduced in Escherichia coli, and the [2Fe-2S] cluster was incorporated into the protein by in Vitro reconstitution. The overall yield was ∼30 mg of purified, reconstituted ferredoxin per liter of culture. Four of the five cysteines in human ferredoxin are coordinated to the iron-sulfur cluster. First, the non-ligand cysteine (cysteine-95) was mutated to alanine, and then double mutants were created in which each of the other four cysteines (at positions 46, 52, 55, and 92) were mutated individually to serine. The wild-type ferredoxin and each of the five mutant proteins were studied by UV-visible spectroscopy and electron paramagnetic resonance spectroscopy. The EPR g av values of all five mutants were very similar to that of wild-type human ferredoxin. In the reduced state, three of the cysteine-to- serine mutants exhibited axial EPR spectra similar to that of wild-type, but one of the double mutants (C52S/C95A) exhibited a rhombic EPR spectrum. The UV-visible spectroscopic properties of the wild- type and the C95A mutant ferredoxins were identical, but those of the other cysteine-to-serine mutant proteins of human ferredoxin were quite different from those of the wild-type protein and each other. These results, along with those from cysteine-to-serine mutations in other ferredoxins, provide the basis for a more comprehensive theoretical and practical understanding of the features important to the ligation of [2Fe-2S] clusters, although they do not yet permit determination of which two cysteines ligate Fe(II) and which ligate Fe(III) in the reduced protein. Ferredoxins are a group proteins of that contain one or more iron-sulfur clusters and participate as electron carriers in biological electron transfer reactions. They display no classical enzymatic functions (Palmer & Reedijk, 1992). Ferredoxins typically are classified by the number and types of iron-sulfur clusters they contain. To date, three types of iron-sulfur clusters in ferredoxins have been identified by X-ray crystallography: [2Fe-2S] (Tsukihara, et al., 1978), [3Fe-4S] (Kissinger, et al., 1991), and [4Fe-4S] (Freer, et al., 1975). Within these categories, ferredoxins can be grouped into families on the basis of their amino acid sequence relationships, reduction potentials, biological source, and spectral properties (Cammack, 1992). For example, the [2Fe-2S] ferredoxins fall into three classes: plant-type, vertebrate-type, and bacterial-type ferredoxins. Plant-type [2Fe-2S] ferredoxins (prototypes include spinach, Anabaena 7120 vegetative and heterocyst, and Spirulina platensis ferredoxins) have reduction potentials around -300 to -460 mV and exhibit rhombic EPR 1 signals with g aV ) 1.96 (g 1 ) 1.88, g 2 ) 1.96, g 3 ) 2.05). Prototypes of vertebrate- type ferredoxins include bovine (adrenodoxin), chick, and human ferredoxins. Putidaredoxin, and the [2Fe-2S] ferre- doxin from Escherichia coli share some of the properties of this group, but may eventually require separate classification. Vertebrate-type ferredoxins show typical axial EPR spectra with g aV ) 1.96 (g 1 ) 1.94, g 2 ) 1.94, g 3 ) 2.02); their reduction potentials are usually near -290 mV, around 200 mV higher than those of the plant-type [2Fe-2S] ferredoxins. The bacterial-type [2Fe-2S] ferredoxin has been isolated only from the nitrogen-fixing saccharolytic anaerobe Clostridium pasteurianum. It is a homodimeric protein with each subunit containing one [2Fe-2S] cluster. The reduced protein shows a rhombic EPR signal with g values of 1.92, 1.95, and 2.00; the reduction potential is about -300 mV. In ferredoxins, the iron-sulfur clusters are bound to the protein by covalent bonds between iron atoms and the sulfur atoms of the thiolate side chains of four cysteine residues. Non-cysteinyl ligands have been reported in other classes of iron-sulfur proteins. For example, the [2Fe-2S] cluster in Rieske-type proteins is coordinated by the sulfur atoms † Supported by National Science Foundation Grant MCB-9215142 and National Institutes of Health Grant GM35752. * To whom correspondence should be addressed. ‡ Graduate Program in Biophysics. § Department of Biochemistry. | Present address: Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. ⊥ Institute for Enzyme Research. X Abstract published in AdVance ACS Abstracts, July 1, 1996. 1 Abbreviations used: EPR, electron paramagnetic resonance; ES- EEM, electron spin echo envelope modulation; HuFd, human ferre- doxin; IPTG, isopropyl thiogalactoside; LB medium, Luria-Bertani medium; MCD, magnetic circular dichroism; NMR, nuclear magnetic resonance; RR, resonance Raman; VFd, Anabaena 7120 vegetative ferredoxin; wt, wild-type. 9488 Biochemistry 1996, 35, 9488-9495 S0006-2960(96)00467-9 CCC: $12.00 © 1996 American Chemical Society