First Working Model Of Cellular Furnace Created

February 14, 1997

Stanford scientists have created the first working model of the cellular furnace that heats and powers nearly all living things - an enzyme called cytochrome c oxidase.

In nature, this enzyme performs the remarkable chemical trick of breaking up tightly bound oxygen molecules and combining them with hydrogen atoms to make water - at temperatures, pressures and acidities compatible with life. The reaction is vital. The energy it releases is used to charge up the cell's biological batteries and to generate heat. Its absence spells death: Cyanide is such a powerful poison because it interferes with cytochrome c oxidase.

Chemists previously have synthesized a number of compounds that structurally resemble this enzyme. But a Stanford research group headed by James P. Collman, the George A. and Hilda M. Daubert Professor of Chemistry, has succeeded in synthesizing a compound that not only resembles cytochrome c oxidase structurally but duplicates its chemical wizardry. The work is reported in the Feb. 14 issue of the journal Science. Other members of the research team are graduate student Lei Fu; Paul C. Herrmann, who is now attending Loma Linda University's medical school; and Xumu Zhang, assistant professor of chemistry at Pennsylvania State University.

The researchers built the functional model to test the current understanding of how this vital biochemical process works. The structure of the enzyme was determined only in the last year, using X-ray crystallography, and many questions remain about how it functions. In the course of making the model, Collman said, he gained some new insights that might help make another long-term scientific dream into a reality - the direct extraction of oxygen from the air without requiring energy-intensive liquefaction.

Cytochrome c oxidase is found in a special structure, called the mitochondria, within the cells of animals, plants and aerobic bacteria, virtually all organisms that use oxygen as an energy source.

The enzyme's chemically active region is made up of two hemes and two copper complexes. A heme is a ring-like chemical structure with an iron atom at its center. Hemes are also found in hemoglobin, which carries oxygen in blood, and myoglobin, which acts as an oxygen store for muscles. Heme is the pigment that gives both blood and muscle tissue their red color.

At the active site of cytochrome c oxidase, one of the hemes and one of the copper complexes form a molecular pocket. Oxygen molecules enter this pocket, bounce around and then attach to the iron atom, and possibly to the copper atom as well.

The metal atoms have a second role as well. They act collectively as a kind of capacitor, storing up an excess charge of four extra electrons. Once the enzyme has the oxygen molecule securely in its grip, it zaps the molecule with its stored electrons. At the same time, positively charged hydrogen ions in the surrounding solution attach to the oxygen atoms. Initially, the hydrogen and oxygen combine to form some very toxic compounds, namely superoxide and hydrogen peroxide. Finally, however, they form water molecules that are released.

"It's a good thing that the enzyme holds onto these intermediate compounds so tightly. They are very nasty actors," Collman said. "For example, Lou Gehrig's disease is associated with the failure of the system that protects the body from superoxide." As far as anyone knows, neither peroxide nor superoxide leak out of cytochrome c oxidase. The superoxide implicated in Lou Gehrig's disease is probably a byproduct of the hemoglobin-based oxygen transport process, he said. The net result of each oxygen-to-water conversion is to push four surplus hydrogen ions through a membrane. This creates a region of elevated positive electrical charge within the mitochondria that causes the hydrogen ions to flow back through the membrane at a different location. The resulting ion flow provides the energy that a second piece of molecular machinery needs to transform adenosine diphosphate (ADP) into adenosine triphosphate (ATP). ATP acts as an energy source for many cellular processes. An active cell needs about two million ATP molecules per second to function.

The model compound that Collman's group synthesized recreates several of the most important features of cytochrome c oxidase's active region. To get a compound that works, however, they had to replace the key iron atom with cobalt. (Their model does not include the second iron atom.) The researchers currently are developing second- and third-generation models that may function without this substitution.

To put the compound to work, the researchers let it soak into a graphite electrode. The electrode is placed into a flask containing water, dissolved oxygen and electrolytic salts. An electric current serves as the source of electrons. By spinning the electrode, the scientists can control the rate at which oxygen flows onto its surface. Their tests show that the model compound successfully converts the oxygen molecules into water.

The researchers point out a number of striking parallels between their analog model and the real thing. Both work by the four electron process. Both use two different metal centers to trap the oxygen molecules and force them to accept additional electrons. Both function at biological pH. Neither leaks hydrogen peroxide.

A key feature in the model came from work that Collman's group performed while modeling the active site of hemoglobin. They successfully synthesized a special chemical superstructure that reduces carbon monoxide's toxicity by interfering with its ability to bind at hemoglobin's active site while not interfering with oxygen's access.

"Zhang, who was a student at the time, realized that our synthetic superstructure could also be used to imitate cytochrome c oxidase. So that's just what we did," Collman said.

The oxygen reduction process catalyzed by cytochrome c oxidase is just one example of a special class of electron transfer reactions that represent a major scientific mystery. These special reactions involve the transfer of multiple electrons to and from small molecules such as hydrogen, oxygen and nitrogen. "These special reactions don't always occur with the speed that they should, theoretically speaking. So, although they can release tremendous amounts of energy, they only occur in the presence of special catalysts," Collman said.

Another biologically important example of these special reactions, which requires the transfer of six electrons, is nitrogen fixation. Bacteria equipped with special enzymes remove nitrogen from the air and convert it to ammonia, which many plants use as food. A major scientific objective is to imitate nature's nitrogen fixation catalysts to provide an inexpensive and environmentally benign source of fertilizer.

"It is our job as bioinorganic chemists to unlock the secrets of these special enzymes," Collman said.

For more information on the Web:

Cytochrome c oxidase home page:

Prof. James P. Collman's home page:

Stanford University

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