Hemocyanin

Uncharted Waters - Investigation of an Oxygen-Transport Protein by Liquid Phase X-Ray Spectroscopy

D. Panzer, M. Hahn, J. Maul and G. Schönhense
Institut für Physik, Universität Mainz, Staudingerweg 7, 55128 Mainz, Germany

C. Beck and H. Decker
Institut für Molekulare Biophysik, Universität Mainz, Jakob Welder Weg 26, 55128 Mainz, Germany

E. F. Aziz
Helmholtz-Zentrum Berlin für Materialien und Energie, Albert-Einstein-Str. 15, 12489 Berlin, Germany

Hemocyanins and oxygen transport

Hemocyanin is a oxygen transport protein similar to the hemoglobin found in the red blood cells of vertebrae like humans. Both are metalloproteins, but where hemoglobin uses iron to bind oxygen during its voyage from the lungs to the tissue, hemocyanin employs copper atoms for this purpose - hence its blue color. It is found in many arthropods (e.g. spiders, scorpions and lobsters) and molluscs (e.g. snails, slugs and squids) and thus the second most popular oxygen transporter in nature. Hemocyanins are not part of blood cells, instead they occur freely dissolved in the hemolymph as an aggregation of subunits, the number and composition depending on the species.

Many hemocyanins from arthropods and mollusks have been investigated extensively - from the genetic level up to the quaternary structure. From X-ray absorption measurements and the few existing crystal structures we already know a lot about the active site of hemocyanin. It is highly conserved between different hemocyanin species and even related proteins like tyrosinase (essential for the tanning process). At the site there are two copper atoms, each coordinated by a set of three histidines that are linked to the protein backbone. This direct link between active site and the protein matrix is necessary for its proper function: When a oxygen molecule is bound between the copper atoms, their distance increases and causes a change in conformation of the whole protein via the aforementioned link. This in turn affects nearby subunits increasing their affinity for oxygen.

oxygenated hemocyanin active site

Figure 1. The active site of hemocyanin in the oxygenated state. The oxygen molecule in the center (white) is bound by two copper atoms (dark) which are in turn coordinated by three histidine residues each. Click here to launch an interactive 3D-model of a complete hemocyanin subunit (Java required).

 

Cooperativity

This change in affinity works in both directions (oxygen uptake and release) and is known as cooperativity. Even though the way it is realized in their structure varies, both hemocyanin and hemoglobin employ this "trick". And it is easy to see why: Without a coordinated effort, binding oxygen in the lungs (high oxygen partial pressure) and releasing it in the tissue material (low oxygen partial pressure) would be rather inefficient. Cooperativity ensures a swift uptake and release of oxygen, therefore making possible the feats of strength and endurance displayed by humans and animals.

Limited access

Investigating a protein like hemocyanin is no easy task in many cases. Since hemocyanins are naturally found in solution, the number of experimental methods is much more limited than with solid samples. It is either necessary to stick to liquid compatible methods (e.g. UV-vis spectroscopy or NMR) or alter the sample in a way that allows for other methods (e.g. freezing, drying) thereby risking unwanted side effects to structure or function.

Shining new light on proteins with soft X-ray spectroscopy

Many aspects of the structural and electronic changes involving the active site have been known for quite some time, but the detailed electronic picture of the oxygenated and deoxygenated states is still sketchy. The two basic states are the oxygenated state with a peroxide bound side-on between two copper II ions, and the deoxygenated without oxygen and a copper valence of I. A newer theory of cooperativity, the so-called nested model, involves four states, two oxygenated and two deoxygenated.

Transition metals like copper are generally well suited for investigation by soft X-ray spectroscopy, as changes in valence, coordination and ligands all affect the probed d-bands. However, soft X-rays necessitate vacuum, and therefore it was impossible for a long time to measure proteins in a liquid this way.

In the past few years, these problems have been overcome by the use of high-speed liquid jets or flow cells equipped with soft X-ray-transparent silicon nitride membrane windows, finally allowing measurements of chemical and biological systems in solution. The apparatus used for our measurements of hemocyanin is called the "Liquidrom", an end-station at the BESSY II synchrotron light source in Berlin.

 

Liquidrom flow setup

Figure 2: Schematic depiction of the Liquidrom.
(a) Flow setup with the liquid circulating behind the membrane.
(b) Static setup with helium atmosphere.

 

Results

As expected, the two basic states show different spectral fingerprints. Some unexpected features in the deoxy spectrum may have their origin in water molecules influencing the active site in the absence of oxygen. For a detailed discussion please refer to our publication:

Water Influences on the Copper Active Site in Hemocyanin
D. Panzer, C. Beck, M. Hahn, J. Maul, G. Schönhense, H. Decker, and E. F. Aziz, Journal of Physical Chemistry Letters 2010 1 (10), 1642-1647. DOI


This work was granted by the Helmholtz-Gemeinschaft via the young investigator fund VH-NG-635, the DFG (H.D., G.S.) and the Center for Computational Sciences in Mainz (H.D.).

 
Weiterführende Links Related Links

 

Last update: Wednesday, 02-Nov-2011 17:09:11 CET Email D. Panzer Impressum