Lincoln, Neb., Oct. 18, 2002 -- A chance decision made by post-doctoral student working with University of Nebraska-Lincoln biochemistry professor Stephen Ragsdale solved a problem Ragsdale had been studying for 22 years.
The decision pointed for the first time to the existence of three metals --nickel, iron and copper --in a protein. The three metals are within a single metal "supercluster." And now that discovery has led to an article in the Oct. 18 issue of Science, the global weekly of research.
Copper was the mystery metal, Ragsdale said. It was unexpected in the crystallized, purified protein distilled from the bacterium Moorella thermoacetica because copper is usually associated with proteins that use oxygen; for this enzyme, oxygen is a poison. Plus, copper inactivates the enzyme when added to it in solution.
"We have known for many years that this 'supercluster' contains nickel that is bridged by some atom(s) we have been calling 'X' to a four-iron cube," Ragsdale said. "My colleagues have asked me for just as many years, 'what is X'?' We never anticipated that X is copper."
In 1985, Ragsdale began trying to crystallize the protein in order to better study it, but, he said, "until 2000, we saw nothing but junk." That year, however, UNL graduate student Tzanko Doukov was "playing with different conditions under which we could get crystals," Ragsdale said, and had a breakthrough. After earning his Ph.D., Doukov left UNL for the Massachusetts Institute of Technology, where he further refined the crystallization procedure so that structure determination became possible.
Doukov's chance decision to use the wavelength for copper as a "blank" to set a benchmark scored a "direct hit," according to Ragsdale, who said he at first thought the result was impossible, but subsequent tests, completed in July in the UNL laboratory, proved that copper was indeed present and essential.
M. thermoacetica is the model organism whose relatives are common anaerobic bacteria found in the guts of many mammals, including humans, Ragsdale said. They are among the most ancient life forms and they use this protein to metabolize carbon monoxide, a characteristic they have retained for 4 billion years, he said.
The bacteria function an as interface between organisms that use simple chemicals such as carbon monoxide, carbon dioxide and hydrogen and more complex organic chemicals. They are the essential underpinnings of the carbon cycle, Ragsdale said, meaning that they take up waste products and convert them for use by the next feeders in the food chain. For example, mammals excrete carbon dioxide (CO2) as they breath; plants take up the CO2 and convert it to their metabolic uses. Animals eat the plants, keeping the cycle going. These particular anaerobes capture and even use carbon monoxide (CO) for energy, keeping it from overwhelming the atmosphere.
"It is interesting that the major toxic effect of carbon monoxide for humans is that it binds to copper in a mitochondrial enzyme preventing oxygen from binding," Ragsdale said. "In this carbon monoxide utilizing enzyme, it is oxygen that is the poison and carbon monoxide is the good guy."
In these organisms, this enzyme even makes carbon monoxide, yet it is never released to its host. The crystal structure also substantiated some biochemical evidence that there is a leak-proof "bottle" within the enzyme that does not allow the CO to escape and ensures that it is used to make its product.
Ragsdale said about half of the known proteins contain metals, but this was the first time these three metals were found together.
He said he had proposed about 20 years ago that the enzyme's metabolic pathway differed from typical metabolic pathways, and coined a term "bioorganometallic reaction sequence," meaning that a series of metal-carbon bonds are formed. One of the highlights of the Science paper is that one of these metal-carbon intermediates was trapped in the crystal structure bound to copper. "Visualizing this intermediate at an atomic level substantiates the concept of a biological organometallic reaction sequence within a metabolic pathway," he said.
He said the race to find this metal chain was internationally competitive in a friendly way.
"I tried to be philosophical about it; that whoever was the first would be adding to knowledge we have and it didn't matter who found it first," he said. "But when you've been working on something for so long, you'd rather not be the second one."
Because the anaerobe's use of the metals is unique and somewhat exotic, Ragsdale said the finding will enrich the biochemical literature and may point to other ways that carbon monoxide, a toxic by-product of combustion, is used by organisms. It also could give clues toward ameliorating growing levels of carbon monoxide in the atmosphere.
The Science article is titled "A Unique Ni-Fe-Cu (nickel- iron-copper) Center in the Crystal Structure of Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Structure." The authors are Doukov, MIT; Tina Iverson, formerly MIT; Javier Seravalli, research assistant professor at UNL; Ragsdale; and Catherine L. Drennan, MIT. The work was funded by the National Institutes of Health and previously by the U.S. Department of Energy.
Contact: Stephen Ragsdale, Professor, Biochemistry, (402)
472-2943 (sragsdale1@unl.edu)
For questions regarding these releases, contact:
tsimons1@unl.edu
(402) 472-8514, Fax: (402) 472-7825