International science team solve biological mystery

Posted by ap507 at Jul 10, 2014 07:10 PM |
Debate over whether a single hydrogen atom exists is finally settled

Issued by the University of Leicester Press Office on 10 July 2014

· Neutron crystallography resolves long-standing molecular mystery

· Research has implications for understanding of oxidative processes within living cells which is crucially important for drug development

· Leicester team worked with colleagues in France, Germany and Manchester

An international team of researchers, led by the University of Leicester, has solved a long-standing mystery in biology, by identifying the molecular structure of a vital biological chemical. The debate – which has raged within the scientific community for years – boils down to something as simple as a hydrogen atom: is it there, or is it not?

The controversy centres around a form of enzyme called a heme (or haem, as in haemoglobin) at the centre of which is an iron atom (Fe) called a ‘ferryl’ which becomes oxidised when a reacting heme is in an intermediate state called Compound I. The question that has taxed biological chemists for decades is whether this oxidation involves just an oxygen atom (O), or a hydroxyl group (OH). The difference being one hydrogen ion, or in other words, a proton.

Much has been written in the scientific literature about this ferryl heme, some scientists arguing that it carries a proton, while others have been equally adamant that no proton is present. Resolving this fundamental inconsistency has implications for understanding of oxidative processes within living cells, which is crucially important for drug development.

Professors Peter Moody and Emma Raven from the University of Leicester, working with neutron crystallography experts from the Institut Laue-Langevin in Grenoble, the Maier-Leibnitz Zentrum(MLZ) near Munich, and the University of Manchester have now solved the mystery of the ferryl heme Compound I structure using a method that has not previously been applied to this problem: neutron crystallography.

“Finding out where all the hydrogens are is key to understanding the way enzymes work,” said Professor Moody. “The ability to capture intermediates at cryogenic temperatures combined with the information available from neutron crystallography, means that we can finally see them.”

Structures of proteins are commonly determined using X-ray crystallography, in which X-rays are fired at a crystalline form of the protein and the resulting diffraction pattern can be used to calculate the molecular structure. While this process works fine in general, it cannot be used to identify the location of hydrogen atoms for various reasons. Neutron protein crystallography, a technique originally developed in the 1960s, uses neutrons instead of X-rays – which eliminates these problems and enables the detection of individual hydrogen atoms. The team used neutron cryo-crystallography, which has the additional complication of using very low temperatures to cryo-trap the enzyme intermediate.

And the answer turns out to be… the ferryl heme in Compound I is NOT protonated. But, unexpectedly, the results showed that one of the amino acid side chains (a histidine) on the molecule IS doubly protonated, which raises questions of its own in terms of mechanisms for oxygen activation in heme enzymes.

“The exact structure of ferryl heme has been a long and difficult problem for the heme community to resolve,” said Professor Raven. “Through our collaborators in Grenoble and Munich, we have been lucky to have had access to excellent neutron beamlines in Europe, which has meant that we were in an excellent position to try new and different approaches that worked very nicely. The single crystal EPR experiments at Manchester were essential to help us verify the stability of the ferryl species.”

The paper ‘Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase’ has now been published in Science.

The research was supported by The Leverhulme Trust, BBSRC, The Wellcome Trust, Bruker UK Ltd, EU FP7, Institut Laue-Langevin and Heinz Maier-Leibnitz Zentrum.

Ends

Notes to editors:

Table of contents for Science Vol 345 Issue 6193

ADDITIONAL QUOTES:

Matthew Blakeley, LADI-III scientist at Institut Laue-Langevin: “This research marks the first time a trapped enzyme intermediate structure has been determined using neutron cryo-crystallography, shedding light on a well-known but unclear catalytic mechanism. The collaboration between the University of Leicester, the ILL in Grenoble and MLZ in Munich brought together leading European researchers in the field to push the boundaries of the technique. Now we have the ability to collect data at various temperatures this opens the way to address many more complex biological problems using neutron crystallography.”

Cecilia Casadei, Institut Laue-Langevin and University of Leicester: "Cryo-cooling of the large protein crystals that are required in neutron crystallography was traditionally considered a challenge. Our work shows the feasibility of this, enabling the low temperature capture of intermediate species in enzymes' reaction cycles by neutron crystallography. The LADI-III diffractometer (Institut Laue Langevin) and the BioDiff machine (FRM II) exploit the unique capabilities of neutron diffraction for the localization of hydrogen atoms in protein structures, which is so often essential for a full understanding of reaction mechanism and catalytic activity."

Andreas Ostermann, Technische Universität München: "This shows clearly the unique advantages of structure determination with neutrons at low temperatures. Postulated reaction mechanisms can be checked and the location of hydrogen atoms in intermediate states of enzymes can be determined."

Tobias Schrader, Jülich Centre for Neutron Science (JCNS): "Neutron protein crystallography beamlines can now offer the same sample environment as common X-ray protein crystallography beamlines can do. This means that in principle all measurements performed with X-rays can be also performed with neutrons when there is the need to find out about the position of hydrogen atoms, which are not visible with X-rays. This illustrates the complementarity of X-rays and neutrons."

Alistair J. Fielding, EPSRC National EPR service, University of Manchester: “Characterisation of enzymatic intermediates is very challenging and requires a multitude of disciplines. In this seminal study, we displayed a range of biophysical techniques all of which were vital for success.”

Background information

Iron is essential for life. The iron in haemoglobin is encapsulated in an organic molecule called a heme, and the heme itself is all wrapped up in a larger molecule, which is the globin structure (hence the name: hemo-globin). Life on Earth often relies on reacting oxygen with metals, and very commonly with iron in the form of catalytic heme enzymes.

In the body, heme enzymes have lots of different roles, such as in respiration and drug metabolism or in removal of toxins such as hydrogen peroxide. As part of this process, the iron becomes highly oxidised and forms a short-lived ‘ferryl’ species. The question which has taxed biological chemists for decades is whether the ferryl species contains just an oxygen atom (ie. Fe(IV)=O) or whether it is protonated (i.e. Fe(IV)-OH). The difference being just a single hydrogen atom.

The enzyme used in the study was cytochrome c peroxidase (CcP) which was isolated and purified then crystallised and reacted to form its Compound I. Compound I normally has a half-life for decay of a few hours at room temperature, but was kept stable for the three week-long experiment by being cooled to -173° C using supercooled nitrogen. Pre-soaking the crystal in ‘heavy water’ (D2O) replaced some of the hydrogens with the heavier isotope deuterium (2H) to provide clearer results.

The neutron source used for the Compound I structure was the research reactor FRM II at the Technische Universität München and the data was collected on the BioDiff instrument. The Compound I structure could then be compared with that of the resting state of CcP which was solved with data collected on the LADI-III instrument at the Institut Laue-Langevin in Grenoble. Colleagues at the University of Manchester EPR service used electron paramagnetic resonance (EPR) to verify that the intermediate was stable at -173° C over the duration of the experiment.

The Leverhulme Trust

The Leverhulme Trust was established by the Will of William Hesketh Lever, the founder of Lever Brothers. Since 1925 the Trust has provided grants and scholarships for research and education; today it is one of the largest all-subject providers of research funding in the UK, distributing over £60 million a year. For more information, visit www.leverhulme.ac.uk

About BBSRC

The Biotechnology and Biological Sciences Research Council (BBSRC) invests in world-class bioscience research and training on behalf of the UK public. Our aim is to further scientific knowledge, to promote economic growth, wealth and job creation and to improve quality of life in the UK and beyond.

Funded by Government, and with an annual budget of around £467M (2012-2013), we support research and training in universities and strategically funded institutes. BBSRC research and the people we fund are helping society to meet major challenges, including food security, green energy and healthier, longer lives. Our investments underpin important UK economic sectors, such as farming, food, industrial biotechnology and pharmaceuticals.

For more information about BBSRC, our science and our impact see: http://www.bbsrc.ac.uk

For more information about BBSRC strategically funded institutes see: http://www.bbsrc.ac.uk/institutes

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