Understanding the Role of Iron in Biology
Iron is essential for the proper function of numerous biological systems in organisms from bacteria to mammals. In humans, iron is essential for the functioning of the respiratory system. The iron atom in hemoglobin, the protein that carries oxygen in the blood, latches onto an oxygen molecule and carries it from the lungs to other parts of the body. My research into the structure and function of iron atoms in other biological systems may lead to a better understanding of disease mechanisms and medical treatments.
Many biological systems require metals to function properly. The purpose of a metal atom may be structural (i.e. to provide support to the three-dimensional framework of a protein) or catalytic (i.e. to provide electrons to facilitate a chemical reaction). A catalyst is a substance that accelerates a chemical reaction without being permanently affected by that reaction; a "catalytic" protein is commonly called an enzyme. My research focuses on two biological systems that require iron(II) for their catalytic function (the II indicates that the iron has lost two electrons). The first system involves the enzyme phenylalanine hydroxylase (PAH), which is the key enzyme that metabolizes phenylalanine, an amino acid you ingest when you eat protein. When PAH doesn't work properly in humans, phenylalanine and related chemicals build up in the brain, causing the disease phenylketonuria (PKU), which leads to mental retardation. By investigating the role of the iron(II) atom in PAH, I hope to understand exactly how PKU occurs. The second system focuses on a cancer chemotherapy agent, bleomycin (BLM). Cancer arises when cells multiply rapidly, producing a mass of tissue called a tumor. BLM cleaves the chain of chemicals (nucleic acids) in cells that makes up DNA, thereby killing the cells and destroying tumors. By focusing on how BLM uses iron(II) to cleave DNA, I hope to gain insight into how this chemotherapy agent and related drugs work.
The primary tool that I use to investigate these systems is spectroscopy. The specific type of spectroscopy that I employ entails shining light at a substance and observing how much light of a particular wavelength is absorbed by the sample. Different metals in various protein environments absorb different wavelengths of light. The type of iron atom that I work with, non-heme iron(II), absorbs infrared and red light (2000-600 nm wavelengths). The specific pattern of light absorbed, or "fingerprint," indicates how many atoms are bonded to the iron and in what geometry. Atoms that are bonded to a metal are commonly called "ligands." Ligands may be from other parts of the protein or drug structure, solvent atoms such as water, and/or the chemicals that the system acts upon ("substrates").
PAH has three substrates that it acts upon: dioxygen (O2), phenylalanine, and a "helper" molecule called a cofactor. The specific reaction that PAH catalyzes is to break up dioxygen and put one oxygen atom each onto the phenylalanine and cofactor. In my work, I have looked at the spectroscopy of the "resting" enzyme (without any of its substrates present), as well as the enzyme with phenylalanine, with cofactor, and with both phenylalanine and cofactor present in the active site of the protein structure. All samples are prepared without dioxygen. In the first three states (resting, phenylalanine-bound, and cofactor-bound), the iron(II) has six ligands attached to it. Crystallography, a type of spectroscopy that uses x-rays to determine the structure of a protein, has shown that these six ligands are three amino acids from the protein structure and three water molecules. When both phenylalanine and cofactor are present, however, one of those ligands (probably a water) is removed. This loss of a ligand would open up free space on the iron(II) atom for dioxygen to react with it. In this way, I am starting to understand specifically how this enzyme facilitates its reaction. My work is now focused on looking at the spectroscopy of PAH mutants. A protein is composed of a chain of amino acids, and a mutated protein is one in which a particular amino acid of the protein chain is changed into another amino acid. I am specifically looking at mutations that have been shown to cause phenylketonuria in humans. By comparing the structure and geometry of the iron(II) site in the working enzyme to a disease-causing variant, I will be able to provide insight into the disease mechanism at a molecular level.
BLM has only two substrates: DNA and dioxygen. In experiments similar
to those I am doing on PAH, I am looking at the spectroscopy of BLM bound
to DNA. DNA is composed of two chains of nucleic acids that pair up to
form a double helix. The pairing of two nucleic acids within the helix,
one from each strand, is called a "base pair." My experiments
involve samples made with full-length DNA isolated from calf thymus glands
that are thousands of base pairs long, and also with small, synthesized
DNA chains that are 10-40 base pairs long. Like PAH, the resting iron(II)
site of the drug has six ligands; although the identities of the ligands
have not been conclusively determined, at least five of them come from
the drug itself. The spectroscopy of BLM bound to DNA indicates that the
DNA changes the iron(II) site and that these changes depend on the specific
sequence, and possibly length, of the DNA. I am working on understanding
these changes and how they correlate to how BLM cleaves DNA. One possibility
is that the binding of BLM to DNA produces an open spot on the iron(II)
atom at which dioxygen can react, in a mechanism similar to what I've
proposed for PAH. Additionally, in order to understand how the drug structure
contributes to its reactivity, I plan to study derivatives of BLM in which
the drug has been synthetically altered. This may allow researchers to
design more effective chemotherapy drugs.
|Modified 15 January 2003 * Contact Us|