ASU Research E-Magazine: Like Acrobats on a Trapeze

Part of what gives proteins their capacities for work are the metal atoms suspended within them. For example, human hemoglobin holds four iron ions, in a coordinating complex. Oxygen binds to this complex tightly enough for a ride through our bloodstream, but loosely enough to be taken away by needy cells.

There are two types of photosynthesis reaction centers in higher plants. Both Photosystem I and II work in concert. Metal ions are suspended in both classes of reaction centers. The metals are manganese and iron.

The atoms from which they are suspended by chemical bonds are called ligands. The metal ions are said to reside in coordination spheres made of the metal and some of the lighter elements, such as oxygen, nitrogen, carbon, and hydrogen. In photosynthetic organisms, the coordinated metal ions are crucial to the transfer of electrons from one side of the membrane to the other.

Russ LoBrutto runs the Electron Paramagnetic Resonance (EPR) Imaging Facility in the sub-basement of the Life Sciences C-wing at ASU. His doctorate is in biophysics. He has worked in a number of different environments, including a medical school.

Electron Paramagnetic Resonance (EPR) is one way to examine the structures of metal-ligand coordination complexes, and how those structures change during the protein’s functional cycle. If the metal atoms have unpaired electrons in their outermost shells, it is possible to do some trickery to help them give up their secrets.

By aligning the unpaired electrons of the metals with a strong magnetic field, and adding electromagnetic energy with microwaves, LoBrutto causes the proteins to absorb a weak but detectable spectrum of radiation. The spectrum tells him what type of metal ion is present, and what is its coordination environment in the protein.

One of the basic attributes of electron is “spin,” but the name doesn’t quite refer to our usual conception. Around an atom, electrons are said to inhabit “orbitals,” spaces which denote both a fairly distinct place and a certain energy level for the electron (given enough energy, an electron can hop up to a higher orbital).

Spin states are limited to two: “up”, or against the applied magnetic field, and “down,” or with the field. Adding energy to an electron can cause it to flip its spin to the opposite state.

Whereas many EPR facilities can detect only the signals of the electrons flipping their spins, ASU’s facility has an additional capacity. By adding radiofrequency energy to the sample in the microwave cavity, LoBrutto can cause nuclear spins in the protein to flip states as well. These nuclear flips are detected as momentary perturbations of the EPR spectrum. This technique is called Electron-Nuclear Double Resonance, or ENDOR.

The EPR signals from a protein molecule are often hard to interpret because the proteins are randomly oriented in the tube, and each orientation gives a slightly different spectrum. The result of combining all possible orientations can be a featureless blob. But by using ENDOR, the information on ligand identities and distances, which is lost in ordinary EPR due to orientation effects, can frequently be recovered.

LoBrutto also has constructed a special type of EPR spectrometer in which very intense microwave pulses, 10-20 billionths of a second in length, replace the weak, steady microwaves used in ordinary EPR or ENDOR. This method, while tricky, is sometimes the best way to regain lost information on metal ligands in randomly-oriented protein samples.

LoBrutto and five other ASU investigators recently were awarded a large NSF grant that will bring ASU’s facility to the state of the art in pulsed EPR technology. The facility’s growth will help Photosynthesis Center scientists to know more of the internal details of the molecules they are studying, and to better understand exactly how those molecules do their jobs.—John Svetlik