TABLE 2.

Effect of 3-fold pore aspartate residues in the iron release process

Proteina	Initial rate of iron releaseb
	NADH/FMN	Sodium dithionite
	μmols/s
LiDps	0.177 ± 0.010	0.095 ± 0.009
D121N	0.084 ± 0.002	0.067 ± 0.009
D126N	0.084 ± 0.002	0.069 ± 0.007
D130N	0.127 ± 0.004	0.069 ± 0.006
D121N/D126N/D130N	0.095 ± 0.004	0.031 ± 0.004

^a Recombinant L. innocua Dps proteins, produced by site-directed mutagenesis, expressed in E. coli strain as apoproteins (∼1 iron/molecule) were prepared in 100.0 mm MOPS, pH 7.0. Mineral was formed in vitro by the addition of FeSO₄ in 1.0 mm HCl at the 240 irons/assembled protein ratio (see “Experimental Procedures”).

^b Reduction and chelation were triggered by adding 2.5 mm reductants (FMN/NADH or sodium dithionite) and 2.5 mm bipyridyl to solutions of protein with mineral (0.2 μm protein and 48.0 μm iron). Measurement of the change in absorbance, at 522 nm/s (the absorbance maximum for the Fe(II)-bipyridyl complex), reflected the conversion of ferric mineral to Fe(II) in solution in the bipyridyl complex. The initial rates were determined by a linear fit of the data points from 0 to 20 s after mixing, which represents the first phase of iron release; the χ² values for the fittings are all >0.95. The data are averages of five or six measurements using two or three independent protein preparations. The error is presented as the standard deviation. All of the differences between LiDps and the 3-fold pore variants are statistically significant (p values < 0.0003).