Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide

Terrance Leighton,Kijeong Kim,Elizabeth C Theil,Xiaofeng Liu

doi:10.1074/jbc.m601398200

Terrance Leighton, Kijeong Kim + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m601398200

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Abstract

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe(2+)/H(2)O(2)/O(2) reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe(2+)/O(2) kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe(2+) were novel in several ways: 1) Ba Dps2 reactions (Fe(2+)/H(2)O(2)) proceeded via an A(650 nm) intermediate, with similar rates to maxi-ferritins (Fe(2+)/O(2)), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe(2+)/O(2) versus Fe(2+)/O(2) + H(2)O(2)) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe(2+)/H(2)O(2) catalysis, inhibited protein-independent Fe(2+)/H(2)O(2) reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H(2)O(2) (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H(2)O(2) destruction, important in host/pathogen interactions. Destruction of H(2)O(2) by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A(650 nm)) and kinetically to the maxi-ferritin diferric peroxo complex.

Highlights

The location of the catalytic site in the protein nanocage is different as are the active site ligands and the oxidant substrate (2, 3, 22, 26 –28). dps genes occur in organisms inhabiting a wide range of environmental niches, including those comparable with primitive, terrestrial atmospheres in at least 130 bacterial and Archaea species (8, 29)
Comparisons of O2 Oxidants in the Dps Protein Fe2ϩ Oxidation Reaction—To understand the oxidant selectivity in Fe2ϩ oxidation by B. anthracis Dps protein ferroxidase sites, we examined the kinetics with different oxidants and followed the reactions spectroscopically
Dps proteins characterized so far, such as the E. coli and L. innocua Dps proteins, preferentially use H2O2 over O2 as the oxidant; rates with O2 are ϳ100-fold smaller in E. coli Dps proteins and are similar to the catalytically inactive animal L ferritin (2, 26, 28)

Summary

EXPERIMENTAL PROCEDURES

Cloning—To clone the dps genes from B. anthracis dlp-1 (Ba dps1) and dlp-2 (Ba dps2), coding regions for Ba Dps and Ba Dps were amplified from the chromosomal DNA of B. anthracis (Sterne 34-F2) by PCR with the following primers: Ba Dps1F, 5Ј-CAAACACATGAACAAACAAGTAATC;

The abbreviations used are

RESULTS

99 Ϯ 20 130 Ϯ 32

DISCUSSION