Nitric oxide sythase (NOS) is known for it production of the free radical nitric oxide (NO). NO has gained much attention of for its role as a neurotransmitter in the brain, a vasodilator in smooth muscle cells, and a cytotoxic agent that targets tumor cells. Shown below is a schematic of NOS producing NO in smooth muscle cells which causes the muscles to relax and allow more blood flow.
The structure of NOS is a homodimer with each monomer containing an oxygenase domain and a reductase domain. The oxygenase domain contains the binding sites for heme, L-arginine (the substrate) and the cofactor tetrahydrobiopterin (BH4). The reductase domain contains the binding sites for NADPH and flavins. In between the domain lies the calcium/calmodulin complex needed for directly coupling of electrons from the reductase domain to the oxygenase domain. Shown below is the heme, crystal structure of the homodimer of NOS and the two domains.
The role of BH4 is not definitively
clear. It is known to serve as an allosteric cofactor but recent
studies suggest it is also involved in redox chemistry. Learning
the exact role of BH4 in the mechanism of NOS is the main focus of my research.
We will be examining the binding and reactivity of a series of natural
and synthetic biopterins to the enzyme. The binding studies will
involve UV-visible absorption spectroscopy and the reactivity work will
utilize rapid scan stopped flow techniques. Analogues of biopterin,
substituted at positions on the pyrimidine ring, have been chosen to determine
how the electronic structure of the pterin affects NOS enzymatic activity.
Experiments will be done with endothelial oxygenase heme domain NOS, eNOS(HD),
preparations in the presence of two different substrates and one inhibitor.
In order to do the binding studies of the analogues to NOS, the biopterins needed to be reduced by hydrogenation. Structures of the reduced biopterin analogues and the natural cofactor are shown below.
Spectra of 4-methoxy-biopterin after hydrogenation for 60 hrs to 4-methoxy-BH4 are shown below.
There were three students working in the Rogers' lab this summer. Peter Barber and Megann Helton worked on the reduction of the biopterin analogues and Robert Ellington worked on kinetic experiments for freshman labs.