Expression, Purification and Characterization of a Novel Rusticyanin from the Psychrotolerant Acidithiobacillus ferrivorans

Abstract

Rusticyanin plays a crucial role in ferrous oxidation of sulfide minerals during bioleaching for industrial metal extraction. Diverse isoforms of rusticyanin have been found, but until now, except for type-A rusticyanin, other isoforms or sources of rusticyanin have been scarcely investigated. Here, a rusticyanin (gene locus 0470) from the psychrophilic Acidithiobacillus ferrivorans was gene-cloned, expressed, purified, and assembled in vitro. All forms of the protein exhibit extreme acid stability, even at pH 0.3. The stability of the protein is obviously enhanced after binding of the copper cofactor; the oxidation state is more stable than the reduced state. The protein has characteristic UV-vis peaks and EPR signals similar to type-A or type-B rusticyanin but is different with a small position shift and an obvious intensity change. The vibrational spectrum of the apoprotein was more different than these between the oxidation and reduced states of the protein. The ferrous oxidation kinetic rate constant of the protein is obviously faster than that of both the type-A and type-B rusticyanins previously reported. Further bioinformatics analysis reveal their changes in sequence and molecule: the mutations related to the peculiar shield belt mostly account for the variation in the properties of the protein, and the classification of the protein as a new isoform, type-C rusticyanin, is proposed.

Purification, Characterization, and Ferrous Oxidation Kinetics of Iron Oxidase from Acidithiobacillus ferridurans

“…The second-order rate constant of the ferrous ion by the oxidation of the iron oxidase in our determination was about 2-3 times faster than those of the reported Chromatium HiPIP, although they were in different solution conditions and reaction directions. Moreover, the kinetic rate of ferrous oxidation by the protein is also obviously faster than that of rusticyanins previously reported [39]. Therefore, our result well supports the view that the iron oxidase plays a role as the initial electron receptor from the ferrous ion in the ferrous biooxidation of the microorganism.…”

“…Our previous study of rusticyanin also exhibited similar shapes and characteristic absorptions [39]. The most obvious difference between the spectra of the oxidized and reduced iron oxidase is in the region range from 1450 to 400 cm −1 , which can be taken as a characteristic fingerprint of conformational change to define the transient states of the protein [39]. A previous study of Clostridial ferredoxin reported that the infrared absorbance peak at 1050 cm −1 , which was quite prominent in native ferredoxin, was absent in apoferredoxin [32].…”

“…The spectra show a common shape of protein spectrum with the characteristic absorption bands such as amide A, B, I, II, and so on, and are nearly same in the amide I and II bands, indicating that the change between the oxidized and reduced states of this protein hardly alters the secondary-structure conformation of the protein. Our previous study of rusticyanin also exhibited similar shapes and characteristic absorptions [39]. The most obvious difference between the spectra of the oxidized and reduced iron oxidase is in the region range from 1450 to 400 cm −1 , which can be taken as a characteristic fingerprint of conformational change to define the transient states of the protein [39].…”

“…The expression of the protein can be performed in a yield with a broader range of conditions than those of the previously reported recombinant Iro protein from A. ferrooxidans Fe1 [32,39] and HiPIPs from various sources [19,20]. The difference between them is that the construction of the expression system of our protein adopted a strategy of codon optimization and gene synthesis, while the encoding genes of the previously reported proteins were all directly cloned from the genomes of their biological sources.…”

“…The iron and sulfide content assays and the MALDI-TOF-MS analysis confirmed that the [Fe 4 S 4 ] cluster of the protein was well assembled into protein even when the protein was produced in E. coli. Although E. coli is a neutrophile and encodes no apparent HiPIPs protein, previously reported active HiPIPs from acidophilic A. ferrooxidans [19] and thermophilic A. caldus [39] were successfully generated in this bacterium, and the iron-sulfur cluster can be correctly assembled and ligated into the binding site of the protein [19]. This protein in an acid solution is more stable than that in neutral solution, which is rightly in line with the extremely acidic periplasm environment that the protein is physiologically located in.…”