Synthesis, Cloning and Expression of a Synthetic Gene for High Potential Iron Protein from Chromatium vinosum

Agarwal, Anshu; Tan, Jing-He; Eren, Mesut; Tevelev, Anton; Lui, Siu Man; Cowan, J. A.

doi:10.1006/bbrc.1993.2626

Cited by 25 publications

(38 citation statements)

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“…5). Because recombinant HiPIPs produced in E. coli exhibit properties very similar to the native proteins (Agarwal et al, 1993;Eltis et al, 1994;Brüser et al, 1998;Caspersen et al, 2000), the described properties of the recombinant Iro 33020 -HisTag produced in E. coli likely reflect those of the native Iro 33020 protein. Indeed, they are similar to those of Iro Fe-1 and Iro BRGM (Fukumori et al, 1988;Yamanaka & Fukumori, 1995;Cavazza et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

et al. 2005

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The gene encoding a putative high-potential iron-sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron-sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a DtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria. INTRODUCTIONOne of the most studied bacteria that thrives in acidic mine drainage is Acidithiobacillus ferrooxidans, formerly Thiobacillus ferrooxidans (Kelly & Wood, 2000;Leduc & Ferroni, 1994). It is characterized as a Gram-negative, acidophilic chemolithoautotroph that obtains energy for its growth mainly from the oxidation of ferrous iron (Fe 2+ ) or reduced sulfur compounds (Ingledew, 1982). Attempts to understand such an energetic metabolism, and especially the oxidation of Fe 2+ , have been initiated, leading to the proposition of several respiratory chains involving various redox proteins that have been identified and characterized in A. ferrooxidans (Fukumori et al., 1988;Blake & Shute, 1994;Yamanaka & Fukumori, 1995;Giudici-Orticoni et al., 1997Appia-Ayme, 1998;Appia-Ayme et al., 1999). Among them, a high-potential iron-sulfur protein (HiPIP) designated Iro, for iron-oxidizing enzyme, was characterized in A. ferrooxidans strains Fe-1 (Fukumori et al., 1988;Kusano et al., 1992) and BRGM (Cavazza et al., 1995) and the corresponding iro gene was also cloned and studied from strain Fe-1 (Kusano et al., 1992). The Iro protein was proposed to be the first electron acceptor in several alternative models of electron transfer chain between Fe 2+ and oxygen (Fukumori et al., 1988;Yamanaka et al., 1995). Based on genetic and subcellular localization studies with strain ATCC 33020, we proposed a model, not involving Iro, in which the electrons are passed from Fe 2+ to oxygen through a set of redox proteins expressed from an operon (Appia-Ayme, 1998;Appia-Ayme et al., 1998, 1999 Bengrine et al., 1998; Yarzábal et al., 2002) that is induced in Fe 2+ -grown cells (Yarzábal et al., 2004). To investigate the possible role of Iro, we probed for the presence of the iro Abbr...

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Section: Discussionmentioning

confidence: 99%

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

et al. 2005

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“…An important goal of these studies is elucidation of the structural and stereoelectronic factors that regulate the electrochemical potential of the cluster. To extend this work to systematic structure-function studies, we recently reported an efficient expression system for Chromatium vinosum HiPIP (8).…”

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“…Sci. USA 92 (1995) 9441 gene region for C. vinosum HiPIP was constructed and efficiently overexpressed in E. coli strain BL21 (DE3) plys S as described elsewhere (8). Mutagenesis was performed according to the method of Kunkel et al (13).…”

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Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

Agarwal

Cowan

1995

Proc. Natl. Acad. Sci. U.S.A.

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The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by sitedirected mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of theThe field of iron-sulfur cluster biochemistry has been revitalized by the important discovery that this class of metalloprosthetic center may also play an active role in nonredox enzymatic chemistry, as exemplified by the centers in Fehydrogenase, aconitase, and endonuclease III (1-3). Extensive studies of model iron-sulfur cluster complexes, native and mutant ferredoxins, and high-potential iron proteins (HiPIPs) have greatly advanced the understanding of cluster chemistry in proteins (4-7). An important goal of these studies is elucidation of the structural and stereoelectronic factors that regulate the electrochemical potential of the cluster. To extend this work to systematic structure-function studies, we recently reported an efficient expression system for Chromatium vinosum HiPIP (8).Previously it has been suggested that the reduction potential of protein-bound [Fe4S4] centers is modulated by the hydrogen bonding pattern to the cluster (9, 10). Recently, however, the role of hydrogen bonding in modulating reduction potentials has been questioned (4, 7, 10). Rather, the number and orientations of hydrophobic residues that surround the cluster (Fig. 1), solvent accessibility, and the influence of the proteinderived electrostatic field have been suggested to play a major role (4, 7). Warshel, Stephens, and co-workers have addressed this issue by a rigorous computational approach (4, 7). Their results support the idea that neither hydrogen bonding nor neighboring ionized and protonated residues significantly influence cluster reduction potentials. Rather, Langevin dipoles from local backbone amides (not necessarily those involved in hydrogen bonding) appear to play a dominant role, while a significant influence from solvent dipoles has also been proposed (7). To address these issues we have embarked on a systematic program to evaluate the functional role of each of the hydrophobic residues surrounding the cluster core of C. vinosum HiPIP. Our initial studies have focused on a conserved residue that lies in close proximity to the reduced (Fig. 1) (11) and has been implicated in defining the cluster potential and electron-transfer pathways. Surprisingly, we find the hypothesis that Tyr-19 modulates the electronic properties of the cluster to be unfounded. In t...

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“…Synthetic genes encoding two HiPIPs have been expressed (Agarwal et al, 1993;Eltis et al, 1994) and are being used to provide such evidence (Lui & Cowan, 1994;Babini et al, in prep.). In this context, we investigated the role of tyrosine-12 in Ectothiorhodospira halophila HiPIP-I (Fig.…”

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The role of a conserved tyrosine residue in high‐potential iron sulfur proteins

et al. 1995

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Conserved tyrosine-12 of Ectofhiorhodospira hdophikz high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y 12H), tryptophan (Y 12W), isoleucine (Y 12I), and alanine (Y 12A). Variants Y 12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y 12F, Y12H, and Y 12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-1 1 .O. Characterization of the Y 12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 0 6 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-591 Val-58 peptide bond, thereby perturbing Gly-46. The AAG;j.. of Y12F of 2.3 kcal/mol with respect to rcWT HiPlP (25 "C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y 12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 "C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

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Synthesis, Cloning and Expression of a Synthetic Gene for High Potential Iron Protein from Chromatium vinosum

Cited by 25 publications

References 0 publications

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

The role of a conserved tyrosine residue in high‐potential iron sulfur proteins

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