Zinc and an N‐terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature

Abstract

Ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 has a 36-residue extra domain at its N-terminus and a 67-residue core domain carrying two iron±sulfur clusters. A zinc ion is held at the interface of the two domains through tetrahedral coordination of three histidine residues (26, 219 and 234) and one aspartic acid residue (276) [Fujii, T., Hata, Y., Oozeki, M., Moriyama, H., Wakagi, T., Tanaka, N. & Oshima, T. (1997) Biochemistry 36, 1505±1513]. To elucidate the roles of the novel zinc ion and the extra N-terminal domain, a series of truncated mutants was constructed: G1, V12, S17, G23, L31 and V38, which lack residues 0, 11, 16, 22, 30 and 37 starting from the N-terminus, respectively. A mutant with two histidine residues each replaced by an alanine residue, H16A/H19A, was also constructed. All the mutant ferredoxins had two iron±sulfur clusters, while zinc was retained only in G1 and V12. The thermal stability of the proteins was investigated by monitoring A 408 ; the melting temperature (T m ) was <109 8C for the natural ferredoxin, <109 8C for G1, 97.6 8C for V12, 89.0 8C for S17, 89.2 8C for G23, 89.3 8C for L31, 82.1 8C for V38, and 89.4 8C for H16A/H19A. K m and V max values of 2-oxoglutarate:ferredoxin oxidoreductase for natural ferredoxin, G1, S17 and L31 were similar, suggesting that electron-accepting activities were not affected by the deletion. The combination of CD and fluorescent spectroscopic analyses with truncated mutant S17 indicated that not only the clusters but also the secondary and tertiary structures were simultaneously degraded at a T m around 89 8C. These results unequivocally demonstrate that the zinc ion and certain parts, but not all, of the extra sequence stretch in the N-terminal domain are responsible not for function but for thermal stabilization of the molecule.Keywords: archaea; ferredoxin; stability; zinc.Ferredoxin is a small protein widely distributed among all living organisms, with one or more redox center(s) composed of acid-labile sulfur and non-heme iron participating in various oxidation±reduction processes in the cell [1,2]. Archaeal ferredoxins are not classified into a simple`archaeal type', but into different categories based on both the clusters and amino-acid sequences; the ferredoxins from thermoacidophilic archaea belong to the 7Fe-bicluster type, those from anaerobic hyperthermophilic archaea to the 4Fe-monocluster type, those from extremely halophilic archaea to the 2Fe-type, and so on [2,3]. It is, however, striking that they play a common role as electron acceptors in the CoA-dependent oxidative decarboxylation of 2-oxoacids, which is seldom found in eubacteria or eukaryotes [4]. In contrast with the widely found 2-oxoacid dehydrogenase multienzyme complex with NAD(P) as an electron acceptor [5], 2-oxoacid:ferredoxin oxidoreductase (OFOR) is a very simple enzyme comprising one to four subunits, with a total molecular mass of around 103±260 kDa [6,7].The primary structure, comprising 103 amino acid residues, of the ferredoxin from the...

High thermal and chemical stability of Thermus thermophilus seven‐iron ferredoxin

“…The seven‐iron ferredoxin from Sulfolobus sp. strain 7 has a thermal midpoint of 109 °C [28]. FdTt, with its 114 °C thermal midpoint at pH 7, is thus one of the most stable seven‐iron ferredoxins (in fact, among proteins in general) investigated to date.…”

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

“…The zinc atom is responsible for a 9°C increase in T m . It is so tightly bound inside the protein that it cannot be removed without removing the two FeS clusters (191). Thermoactinomyces vulgaris subtilisin-type serine-protease thermitase contains three Ca 2ϩ -binding sites; one of them is not present in its mesophilic homologues (331).…”

Iron-Sulfur World in Aerobic and Hyperthermoacidophilic ArchaeaSulfolobus

“…The sequence comparisons suggest that three histidine residues in the N-terminal extension region and one conserved aspartate in the ferredoxin core fold are strictly conserved in all archaeal zinc-containing ferredoxins [17, 41, 43] (see Figure 1(a)), which suggests that they probably serve as ligands to the isolated zinc center. Although the isolated zinc site apparently contributes in part to ferredoxin thermal stability [50–52], zinc-lacking isoforms, for example, from Sulfolobus metallicus [53] and Acidianus ambivalens [54], have devised a natural strategy that accounts for an enhanced thermal stability without using the zinc site. It remains unknown whether another metal such as iron could replace the mononuclear zinc site of zinc-containing ferredoxin, when the archaeal cells are grown under zinc-limited conditions.…”