Response of Rubredoxin from Pyrococcus furiosus to Environmental Changes: Implications for the Origin of Hyperthermostability

Abstract

The bases of the hyperthennostability of rubredoxin from Py~ococcus furiosus (RdPf) have been probed by structural perturbations induced by solution pH and ionic strength changes. Comparison of the solution behavior at pH 7 and pH 2, as probed by far-and near-UV circular dichroism, Trp fluorescence emission, 1 -anilinonaphthalene-8-sulfonate (ANS) binding, and NMR spectroscopy, reveals the presence of only minimal structural variations at room temperature. At pH 2, the protein displays a surprising nearly native-like behavior at high ionic strength while, at low ionic strength, it is capable of strongly binding the hydrophobic probe ANS. All the secondary and tertiary structural features, including the environment of the hydrophobic core, appear to be intact regardless of pH and ionic strength. The apparent "melting" or denaturation temperature at pH 2, however, is 42 "C lower than at pH 7. This is attributed to the perturbation of many electrostatic interactions, including the disruption of all the ion pairs, which is complete at pH 2, as indicated by electrometric pH titrations. The implications of these findings for the origins of the hyperthennostability of rubredoxin are discussed.The existence of life forms that live at elevated temperature and pressure is an open manifestation of nature's ingenuity in managing to support life even under the most adverse conditions. A variety of so-called hyperthermophilic microorganisms have been isolated in recent years (Stetter et al., 1990;Adams, 1990;Stetter, 1986) in the vicinity of both terrestrial and submarine hydrothermal vents. The majority of them are classified as Archaea (Woese et al., 1990) and thrive at temperatures up to 110 "C, the highest temperature known to be compatible with life. They are also the most slowly evolving or "primitive" of known organisms. Little is known of the novel biochemistry that must be required to sustain life above 100 "C. However, the quest for biomolecules displaying unusual and interesting physicochemical properties has recently led to the discovery and isolation of specific enzymes from these hyperthermophilic organisms. Among those that have been purified so far are proteases (Eggen et al., 1990;Cowan et al., 1987), redox proteins (Aono et al., 1989;Blake et al., 1991), hydrogenases Juszczak et al., 1991;Pihl & Maier, 1991), dehydrogenases (Robb et al., 1992), ferredoxin-dependent oxidoreductases (Mukund & Adams, 1990), DNA polymerases (Lundberg et al., 1991), and enzymes involved in carbohydrate metabolism (Koch & Zablowski, 1990;Constantino et al., 1990). These enzymes are composed of the regular 20 amino acids and display a thermal stability unknown to their mesophilic counterparts. Yet, sequence comparisons reveal no apparent striking differences. At this juncture, the origin of protein hyperthermostability (Flam, * To whom correspondence should be addressed.

Rubredoxin from the Green Sulfur Bacterium Chlorobium tepidum Functions as an Electron Acceptor for Pyruvate Ferredoxin Oxidoreductase

“…EPR and CD Spectra of C. tepidum Rd-The near UV-visible CD spectra of oxidized C. tepidum Rd closely resembled spectra obtained previously (31,45,50), with the exception of the absorption maximum at 385 nm and the shoulder at 420 nm (Fig. 3A).…”

Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes

“…Ion pairing also plays a role in protein stabilization (9). This was also confirmed by the determination of the structure of glutamate dehydrogenase (GDH) from Pyrococcus furiosus, which was compered with GDH from mesophilic origin.…”

Entropic Stabilization of the Tryptophan Synthase α-Subunit from a Hyperthermophile, Pyrococcus furiosus

“…High resolution structures of proteins from hyperthermophiles have shown that the number of ion pairs in most of the hyperthermophile proteins is higher than in mesophile counterparts (4 -13), although additional ion pairs were not observed in some hyperthermophile proteins (25)(26)(27)(28). Table III lists the following structural segments: five intra-helical, three inter-helical, two between helix and loop, one between helix and ␤-strand, one between ␤-strands, and one between ␤-strand and loop (Table III).…”

“…However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability (14 -19), whereas others have shown a favorable contribution (20 -24). There are also reports on some hyperthermophile proteins without additional ion pairs (25)(26)(27)(28). The conformation of general globular proteins is marginally maintained by the combination of many positive (such as hydrophobic interaction and hydrogen bond) and negative (such as entropic effect and steric hindrance) factors for stabilization (29).…”