Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Y A Oñate,S J Vollmer,R L Switzer,M K Johnson

doi:10.1016/s0021-9258(18)51476-9

Abstract

The properties of the [4Fe-4S] cluster in glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis have been investigated using low temperature magnetic circular dichroism, electron paramagnetic resonance (EPR), and resonance Raman spectroscopies. The Raman spectra of the native enzyme in the Fe-S stretching region show a [4Fe-4S]2+ cluster that is structurally very similar to those in simple redox proteins. Photochemical reduction mediated by 5-deazaflavin with oxalate as the electron donor resulted in [4Fe-4S]+ clusters with a mixture of ground state spin multiplicities. Magnetic circular dichroism and EPR studies of samples ranging in concentration from 0.15 to 0.4 mM concur in finding S = 3/2 [4Fe-4S]+ clusters with predominantly axial and positive zero field splitting as the dominant species. The EPR studies also revealed minor contributions from S = 1/2 [4Fe-4S]+ centers and an S = 5/2 species. The latter becomes the dominant component in more concentrated samples (approximately 2 mM), and arguments are presented in favor of assignment to S = 5/2 [4Fe-4S]+ clusters rather than adventitiously bound high spin Fe(III) ions. The concentration-dependent spin state heterogeneity of the [4Fe-4S]+ cluster in glutamine phosphoribosylpyrophosphate amidotransferase is discussed in light of the magnetic and electronic properties of the [4Fe-4S]+ centers in other enzymes and proteins.

Highlights

From the $Department of Chemistry and Center for MetalloenzymeStudies, University of Georgia, Athens, Georgia 30602and the §Department of Biochemistry, University of Illinois, Urbanu, Illinois 61801
The amino acid sequence shows 4 cysteinyl perature magnetic circular dichroism, electron para- residues in the COOH-terminal segment with an arrangement magnetic resonance (EPR), and resonance Raman characteristic for [4Fe-4S] clusters of the ferredoxin type [5]
Magnetic circular dichroism and EPRstudies of samples ranging in concen

Summary

Introduction

From the $Department of Chemistry and Center for MetalloenzymeStudies, University of Georgia, Athens, Georgia 30602and the §Department of Biochemistry, University of Illinois, Urbanu, Illinois 61801. The ization of the oxidized ( n = 2) and reduced ( n = 1) forms of latter becomes the dominant component in more con- the [4Fe-4SIn+cluster in B. subtilis amidotransferase using centrated samples (-2 mM), and arguments are pre- low temperature EPR, MCD,' and resonance Raman specsented in favor of assignment to S = 512 [4Fe-4S]+ troscopies.

Results

Conclusion