A sulfite respiration pathway from Thermus thermophilus and the key role of newly identified cytochrome c₅₅₀

Abstract

Sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. Various detoxification mechanisms exist, but sulfite oxidoreductases (SORs) are one of the major actors in sulfite remediation in bacteria and animals. Here we describe the existence of an operon in the extreme thermophilic bacterium Thermus thermophilus HB8 encoding both a SOR and a diheme c-type cytochrome. The in vitro analysis clearly showed that the newly identified cytochrome c₅₅₀ acts as an acceptor of the electrons generated by the SOR enzyme during the oxidation of sulfite. The electrons are then rapidly shuttled via cytochrome c₅₅₂ to the terminal ba₃- and caa₃-type oxidases, thereby unveiling a novel electron transfer pathway, linking sulfite oxidation to oxygen reduction in T. thermophilus: sulfite → SOR(HB8) → cytochrome c₅₅₀ → cytochrome c₅₅₂ → ba₃ oxidase/caa₃ oxidase → O₂. The description of the complete pathway reveals that electrons generated during sulfite oxidation by the SOR are funneled into the respiratory chain, participating in the energy production of T. thermophilus.

Fig. 1.

Genomic arrangement of the putative SOR genes in T. thermophilus HB8 and multiple sequence alignment of cytochromes c₅₅₀. (A) The presence of a promoter and a terminator putative region suggests that the genes form an operon. Promoter, predicted −35 and −10 regions as well as the start of transcription (shown in bold). Terminator, U-rich region (depicted in bold); the predicted hairpin loop is shown. (B) Multiple sequence alignment of the cytochrome c₅₅₀ from T. thermophilus HB8 and HB27, the cytochrome c₅₅₂ from T. thermophilus HB8, and the sulfite:cytochrome c oxidoreductase subunit B Cj0004c from C. jejuni NCTC 11168 (CC). The predicted leader sequence is underlined. Residues determined by internal sequencing are shown in bold. Residues of the heme-binding sites are indicated by an asterisk, while methionine residues potentially involved in heme coordination are indicated by triangles.

Fig. 2.

Reduction of cytochrome c₅₅₀ by SOR_HB8 in the presence of sulfite. Absorption changes collected after anaerobically mixing oxidized cytochrome c₅₅₀ (1 μM) with 2 mM sulfite in the absence (dashed line) or in the presence (continuous line) of ∼6 μM SOR_HB8. Temperature, 45°C.

Fig. 3.

Cytochrome c₅₅₂ mediates cytochrome c₅₅₀ oxidation by ba₃ or caa₃ oxidase, as shown in the time course of cytochrome c₅₅₀ oxidation by ba₃ (A) or caa₃ (B) oxidase in the absence (dashed traces) or presence (solid traces) of increasing concentrations of cytochrome c₅₅₂. Temperature, 25°C; λ, 418 nm. Prior to the experiment, cytochrome c₅₅₀ was reduced with 300 μM ascorbate. Concentrations after mixing: [c₅₅₀] = 500 nM; [ba₃] and [caa₃] = 100 nM; [c₅₅₂] = 0, 0.2, 0.4, 1.0, 2.0, 5.0, and 10 nM (A) or 0, 0.2, 0.4, 1.0, 2.0, and 5.0 nM (B), from right to left. (Insets) Linear dependence on cytochrome c₅₅₂ concentration of the reciprocal of the reaction half-time.

Fig. 4.

Kinetics of electron transfer from cytochrome c₅₅₀ to cytochrome c₅₅₂. (A) Absorption changes measured at 416 nm after mixing 5.5 μM oxidized cytochrome c₅₅₂ with 3.4 μM cytochrome c₅₅₀ prereduced with 1 mM ascorbate. Temperature, 4.5°C. Ionic strength, 12 mM. The experimental trace (dashed) is shown together with its best fit (solid line) (see Materials and Methods). (Inset) Ionic strength dependence of the observed rate constant. (B) Observed rate constant measured as a function of cytochrome c₅₅₀ concentration at [c₅₅₂] = 2.75 μM (•) or 2.05 μM (○). Second-order rate constants estimated according to the methods described in reference : k_F = 5.5 × 10⁷ M⁻¹ s⁻¹ and k_R = 0.5 × 10⁷ M⁻¹ s⁻¹ at the lower ionic strength (•); k_F = 2.0 × 10⁷ M⁻¹ s⁻¹ and k_R = 0.2 × 10⁷ M⁻¹ s⁻¹ at the higher ionic strength (○).

Fig. 5.

Sulfite respiration. O₂ consumption by ba₃ cytochrome oxidase in the presence of SOR and the cytochromes c₅₅₀ and c₅₅₂, with sulfite serving as the primary electron donor. Values refer to the observed rate of O₂ consumption, expressed in μM O₂ min⁻¹. Concentrations: [sulfite] = 2 mM; [c₅₅₂] and [c₅₅₀] = 0.5 μM; [ba₃] = 0.3 μM; [Asc] (ascorbate) = 6.7 mM; [TMPD] = 0.2 mM. In trace A, the two SOR additions were 1.8 μM and 3.6 μM, respectively; in trace B, 5.6 μM SOR was present. Temperature, 37°C.

Fig. 6.

Proposed mechanism of sulfite metabolism via the SOR system in T. thermophilus. Electrons generated during sulfite oxidation are captured by cytochrome c₅₅₀ and transferred to the terminal oxidases via cytochrome c₅₅₂ to finally reach the ultimate acceptor, molecular oxygen. The electron transfer from the bc₁ complex to the terminal oxidases via cytochrome c₅₅₂ is also illustrated. PDB IDs: cytochrome c oxidase ba₃, 1EHK; cytochrome c₅₅₂, 1C52.