0162-0134/00/$ - see front matter q2000 Elsevier Science Inc. All rights reserved. PII S0162-0134 ( 99 ) 00202-0 Monday Jan 31 02:44 PM StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry) Article: 6298 www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 78 (2000) 35–41 Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens Pernilla Wittung-Stafshede a, *, Claudio M. Gomes b , Miguel Teixeira b ´ a Chemistry Department, Tulane University, New Orleans, LA 70118, USA b Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa, Apt 127, 2780-156 Oeiras, Portugal ´ ´ Received 11 August 1999; received in revised form 11 October 1999; accepted 21 October 1999 Abstract The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric protein containing two iron–sulfur centres, one [3Fe–4S] 1q/0 and one [4Fe–4S] 2q/1q . It is an intrinsically hyperstable protein, being expressed at the organism’s extreme optimal growth temperature: 80 8C. Using spectroscopic methods we have investigated the unfolding reaction of the Acidianus ambivalens ferredoxin. No unfolding of the oxidised ferredoxin was observed at pH 7.0, even in the presence of 8 M GuHCl. Upon increasing the pH to 10.0, the unfolding transition showed a midpoint at 6.3 M GuHCl and an unfolding-free energy of 70 kJ mol y1 in buffer (pH 10) was estimated. Kinetic-unfolding experiments showed that the polypeptide unfolding correlated with rearrangement of the iron–sulfur centres to new ones which had strong absorption maxima at 520 and 610 nm. These new, possibly linear three-iron, clusters were coordinated to the unfolded protein but degraded slowly. From thermal experiments in the presence of GuHCl we estimated the melting temperature for the Acidianus ambivalens ferredoxin in buffer (at pH 7) to be 122 8C. Possible structural properties that contribute to the large thermal stability of the Acidianus ambivalens ferredoxin are discussed using a three-dimensional protein model. q2000 Elsevier Science Inc. All rights reserved. Keywords: Ferredoxin; Thermostability; Archaea; Protein folding; Spectroscopy 1. Introduction 1.1. Thermostable proteins Most soluble proteins are minimally stable, exhibiting a small difference in stabilisation between the native (folded) and unfolded forms (around 30–50 kJ mol y1 ). This stabilis- ation energy has been attributed to the cumulative effect of hydrogen bonds, ion pairs and hydrophobic interactions [1,2]. Some proteins show enhanced thermal stability but do not exhibit significant differences in their stabilisation ener- gies [2]. A large number of mechanisms are thought to be responsible for determining such hyperstability of proteins. The elucidation of these, to date mostly unknown, mecha- nisms is essential to obtain an understanding of protein sta- bility and folding. In the pursuit of this goal, the study of proteins and enzymes isolated from extremophiles, i.e., microorganisms which are capable of thriving in environ- ments in which some physical conditions such as temperature, salinity and pH are at their limits, and a comparison to equiv- alent proteins from non-extremophiles have been undertaken. * Corresponding author. Tel.: q1-504-862-8943, fax: q1-504-865-5596; e-mail: pernilla@mailhost.tcs.tulane.edu Proteins isolated from (hyper)thermophilic organisms, which are intrinsically stable at the optimal growth temper- atures of the organisms from which they are isolated (above 55 8C) are good models for addressing the nature of protein stability. In the last few years an extensive sequence of struc- tural and biochemical data has been accumulating (e.g. [3– 5]), thus allowing comparative studies with homologous proteins isolated from mesophilic sources. In particular, enzymes have been extensively studied due to their potential biotechnological interest. Table 1 compiles a few selected thermophilic enzymes, which are given as examples of inten- sively studied enzymes (see e.g. [3,4]) with respect to ther- mostability determinants, and for which a crystallographic structure is available. A more comprehensive list of thermo- philic proteins and their properties, but without structural data, can be found in [3]. From all the data available at this point, it is clear that no general strategy accounting for protein thermal stability can be established [1,2,5]. What is generally observed in ther- mophilic proteins is that: (i) they are strikingly similar to their mesophilic counterparts in terms of topology and enzy- matic mechanisms; (ii) there is no generalised correlation between the amino acid composition and thermal stability;