Novel protein complex enables survival in hostile environment

Biswarup Mukhopadhyay and Eric Johnson from the Virginia Bioinformatics Institute at Virginia Tech have discovered a novel enzyme that represents an ancient detoxification system and provides a clue to the development of early metabolism on earth.

The research appears in the Nov. 18, 2005 issue of the Journal of Biological Chemistry, in the article “A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the Methanoarchaeon Methanocaldococcus jannaschii”.

“The newly discovered enzyme links biological methanogenesis and sulfate reduction, two most ancient respiratory metabolisms, in a unique way,” said Mukhopadhyay, whose lab studies organisms that produce methane, in particular M. jannaschii.

Commenting on the research, William Whitman, professor of microbiology at the University of Georgia and an expert in microbial diversity and the evolutionary relationships of prokaryotes, said: “This original work provides important insights into the evolution of the methanogens. These organisms have often been thought to be very limited in their metabolic capabilities. The current study goes a long way to dispelling this simplistic view and greatly extends our knowledge of their versatility.”

Methanogenesis is a microbial process in nature that produces methane, an energy resource and a green house gas. Sulfate reduction is also a microbial process where organisms turn sulfate into sulfide, a corrosive compound or gas that smells like rotten eggs.

Methanogenesis is a 2.7–3.2-billion-year-old process and sulfate reduction originated at least 3.7 billion years ago on earth. “These two processes apparently cannot exist within one living cell, because the reduction of sulfate produces sulfite as an intermediate, which damages an essential component of the methane production machinery,” Mukhopadhyay said. “Consequently, sulfite kills most methanogens.”

However, early methanogens must have been able to tolerate sulfite. “Early earth had a lot of sulfide but no oxygen until about 2.7 billion years ago. Then, the reaction of the small amounts of oxygen with sulfide would have produced an incomplete oxidation product – sulfite,” he said. “Methanogens present during the oxygenation of earth had to face this sulfite.”

But Johnson and Mukhopadhyay could not find any sign of such ability in the DNA sequence data for methanogens. “It was clear that either the ancient sulfite detoxification has been lost or it is not recognizable because it is unlike any known system,” Mukhopadhyay said.

The challenge of the latter possibility attracted the group to the topic. They decided to see if methanogens that live in an environment where the early earth conditions are preserved – deep-sea hydrothermal vents – still have the ancient detoxification system.

Inside a hydrothermal vent, sulfide-containing superheated water at 350 C (662 F) mixes with cold oxygen-containing water, creating cooler environments — 48 to 94 C (118 to 200 F) — where M. jannaschii can thrive. “This sulfide-oxygen mixture can also generate sulfite. Therefore, M. jannaschii experiences conditions that existed on early earth,” Mukhopadhyay said.

He knew that Lacy Daniels, his mentor at the University of Iowa, and Negash Belay, a colleague during his graduate studies, had found sulfite assimilation ability in an organism closely related to M. jannaschii, but had not investigated how that organism handled the sulfite toxicity. Putting all these pieces of information together, Johnson and Mukhopadhyay hypothesized that M. jannaschii has a sulfite-reducing enzyme and began to search for this system.

Protein analysis of M. jannaschii from sulfite-free and sulfite-enhanced environments revealed that M. jannaschii tolerates sulfite and even uses it as a sulfur source by expressing an enzyme not seen previously. The enzyme, which is located on the cell membrane, converts toxic sulfite into sulfide, an essential nutrient of M. jannaschii.

This enzyme, coenzyme F420-dependent sulfite reductase, or Fsr, “uses an unusual coenzyme – a deazaflavin molecule called Factor 420 — as an electron carrier for the reduction of sulfite. None of the previously described sulfite reductases use F420,” Johnson said.

By use of genome-sequence-driven proteomics techniques, they identified the gene for the enzyme. A search showed that this gene exists only in hydrothermal vent methanogens and their close relatives, but not in other microorganisms.

From the sequence of the fsr gene, Johnson and Mukhopadhyay discovered that the novel activity of Fsr comes from a unique structure; two previously known proteins with unrelated functions have been physically combined by use of a linker. Even after this linking, the two units retain their individual characteristics.

“We hypothesize that the NH2-terminal half of Fsr (named Fsr-N) collects electrons via F420 and the COOH-terminal half (Fsr-C) uses those electrons to reduce sulfite to sulfide,” Johnson said.

In their experiments, the researchers detected both of these individual properties as well as the combined activity. “Fsr-N resembles a protein that introduces electrons into the membrane-based energy transduction systems of certain archaea. Such an energy transduction system is also found in E. coli and humans,” Mukhopadhyay said. “Fsr-C is similar to the sulfite reductases that are found in certain bacteria and archaea. These previously described sulfite reductases do not use coenzyme F420 as the electron source and are also not tethered to their electron-donating partners.”

“The existence of Fsr poses several questions that are important in the context of evolution of metabolism and enzyme mechanism,” Mukhopadhyay said. “We do not know whether the splitting of the fsr gene gave rise to the sulfite reductases of the bacteria and energy transducers of certain archaea or if this enzyme originated from a gene fusion event.”

“From the affinities and reaction rates it is clear that the enzyme will sense even a minute amount of sulfite and will neutralize even a large amount of sulfite very quickly. These properties suited the need of the ancient methanogens when oxygen appeared on earth,” Mukhopadhyay said. “But, why did the organism have this enzyme in the first place?”

A clue comes from published works by Robert White, professor of biochemistry at Virginia Tech, who studies how metabolic systems evolved and collaborates with Mukhopadhyay. “It is possible that M. jannaschii had this enzyme for cofactor biosynthesis and having it in advance gave the organism a selective advantage when oxygen, and consequently sulfite, appeared,” Mukhopadhyay said. “Since we now know that methanogens had a way to handle sulfite toxicity, we could hypothesize that the rest of the sulfate reduction pathway once existed in these organisms.”

Johnson and Mukhopadhyay have already seen some remnants of this system in M. jannaschii. Thus, they say, it is possible that methanogenesis and sulfate reduction could have originated in the same organism after all, and, in the course of time, a loss of the sulfite reductase gene gave rise to a sulfite-sensitive methanogen. Similarly the loss of certain key genes gave rise to the archaea that reduces sulfate, but do not make methane. “But it is equally possible that the sulfite reduction system was developed in another organism and the methanogens acquired the sulfite reduction gene via horizontal transfer from that entity,” Mukhopadhyay said.

Rolf Thauer, professor and head of the Department of Biochemistry at the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany, and a noted authority on anaerobic microorganisms, commented: “The finding of a novel sulfite reductase in a methanogenic archaeon is an important discovery. It may prove to be directly relevant to the anaerobic oxidation of methane with sulfate, a process in which archaea closely related to methanogenic archaea are intimately involved.”