Channel that transfers and protects the heme
Cofactor and prosthetic groups: A cofactor is a non-protein compound that is bound to a protein generally an enzyme and is needed for the protein’s biological activity, for example helps the enzyme in biochemical transformations. Cofactors can be tightly or loosely-bound to an enzyme. Loosely-bound cofactors are termed coenzymes and tightly-bound cofactors termed prosthetic groups. Proteins that contain a metal ion cofactor are called metalloproteins.
What is heme?
Heme is a member of a family of compounds called porphyrins (a group of organic compounds which many occur in nature, e.g., pigment in red blood cells). It is a prosthetic group that consists of an iron atom contained in the center of a large porphyrin ring. Metalloproteins that have heme as their cofactor are called heme proteins.
Heme is made inside a cell organelle. Synthesis of heme occurs in the mitochondria and cytoplasm of a cell. The process begins in the mitochondria, the final step (which produces the heme) is also mitochondrial but all intermediate steps are cytoplasmic.
Heme plays a crucial role in supplying a cell with the energy needed to carry out the chemical reactions that sustain life. More than half the heme in the body is in hemoglobin, but some of the rest is in the heme protein called Cytochrome c, or cyt c that is found loosely associated with the inner membrane of the mitochondrion. Cytochrome c taps energy out of food.
The iron atom in the heme serves as a source or sink of electrons. (Iron serves numerous important functions in the body relating to the metabolism of oxygen, for e.g. its role in transport of oxygen by hemoglobin.) Once heme is made inside a cell organelle it must be moved outside and plugged into a protein before it becomes functional. While moving across the cell membrane heme must be protected as it is chemically vulnerable because of the iron atom. A channel is required to both transfer and protect the heme.
Work done by Robert Kranz and Elaine Frawley
Scientists at Washington University in St. Louis discovered a channel present in plants and many bacteria that both transfers and protects the heme. The discovery was made by biology professor Robert Kranz and graduate student Elaine Frawley.
According to Kranz who has devoted much of his career to understanding the molecular machinery that makes cytochrome c, the heme group is always assembled in the protected environment inside a cell organelle or bacterium, and then moved outside, where it is locked into a cytochrome c protein sitting on the outside of the cell membrane.
To figure out how heme gets across the membrane, Frawley used a benign strain of common gut bacterium Escherichia coli. This bacterium was a special E. coli whose own cytochrome-c making machinery had been removed and replaced with machinery taken from Helicobacter hepaticus, a recently discovered bacterium that can cause hepatitis.
The Helicobacter system for making cytochrome features a humongous protein called CcsBA that threads repeatedly through the cell membrane, leaving messy loops on either side like the stitches made by a sewing machine whose tension is out of adjustment. The parts of the molecule that pass through membrane are called transmembrane domains. By trapping the heme inside the isolated channel protein, Frawley showed that two transmembrane domains come together to form a channel for the heme.
Kranz explains proteins like CcsBA that are embedded in the cell membrane are difficult to work with. The membrane is a lipid, or fat, so trying to extract a membrane protein is like trying to wash greasy dishes in cold water. Frawley had to use detergents to separate the protein, he says, and even then it took her a year or two to purify enough protein to work with.
Frawley says, “When I got enough pure protein and concentrated it in the test tube, I could see it was tinted red, the color of heme. That told me I had trapped the heme in the channel protein. I was ecstatic!” To be sure Frawley analyzed the sample spectroscopically and confirmed that it was indeed absorbing light at the wavelengths characteristic of heme.
How is heme protected in transit?
Kranz and Frawley knew that some parts of the membrane protein CcsBA are highly conserved, meaning that no matter how evolution alters the rest of the protein, it leaves these sections alone. They suspected the conserved bits, four copies of the amino acid histidine, are guard molecules that protect the heme from oxidizing. When Frawley mutated the histidines on the inside of the membrane, the protein’s absorption spectrum told her it had stopped functioning. The heme channel protein couldn’t bind the heme and shuttle it through to the outside anymore. “That’s the key point of the paper,” says Kranz. “There’s a heme channel and the histidines have to be there to both bind heme and protect the heme from the environment.”
To prove the mutated histidines were the problem, Frawley added imidazole, a small compounds that is chemically the same as part of the histidine, to her E. coli cultures. The imidazole fixed the broken channel and the E. coli started making cytochrome again.
The imidazole trick, Kranz says, “is probably the coolest result I’ve had in 23 years in my lab.”
Source: http://news-info.wustl.edu/news/page/normal/15249.html
December 18, 2009