Roger Thorneley and David Lowe

THE FOUNDATIONS OF THE LT MECHANISM FOR NITROGENASE

In nineteen eighty-four, a date that resonates in more than one way, David Lowe and Roger Thorneley published four back-to-back papers in The Biochemical Journal (1-4). Insofar as they contained the same title and the same authors – “The mechanism of Klebsiella pneumonia nitrogenase action” – all the papers were equal. They described the kinetic transformations involved in the catalytic mechanism of the monstrously complicated nitrogenase enzyme; and, they laid the foundations for much of the work that has followed. On a fresh, windy, but sunny day at the RSC EPR meeting in Colchester more than thirty years later, we meet to discuss the origins of the now so- called Lowe-Thorneley (LT) mechanism. I first met Roger at an IBDG meeting in about 1995; David a few years later. I haven’t seen either of them in a good many years. But they are instantlyrecognizable from across the room. So far as I wasever concerned, they both always looked about 60; and they still do. It’s as if I am meeting two brothers, so alike are they and so familiar with one another’s foibles. Figure 1 demonstrates an early likeness. Their relationship goes back even further than one might imagine. Their mothers went to the same school and, as a schoolboy, David met Roger’s Great Uncle Arnold – who was Director of the Ceramic Research Institute, Stoke – on Trent – on more than one occasion as Uncle Arnold would appear annually to present the school prizes (and is rumoured to have said: “Not you again, Lowe”).

Lowe and Thorneley

Figure 1: Those were the days. Lowe (left) and Thorneley (right), at the Unit for Nitrogen Fixation, Sussex. In the background is Paul Maryan.

A scientific melting pot at Sussex
Their scientific paths crossed at the Unit for Nitrogen Fixation (UNF) in Sussex. The UNF was set up in 1965 after it moved from Queen Mary, with Joseph Chatt as the director. Chatt was an organometallic chemist and the philosophy at UNF was to have chemistry and microbial work (with John Postgate) side by side (see (5) for a more detailed history). The UNF included well-known names such as Ray Dixon, Howard Dalton, Geoff Leigh and Michael Mingos, Bob Crabtree, John Dilworth, and Ray Richards. The scientific mission was simple: to understand nitrogen fixation. The initial approach was to use transition metal complexes, and in parallel to employ microbiology methods (because only bacteria fix nitrogen), but the Unit later expanded to include protein chemistry which included Bob Eady and Barry Smith. Roger, with expertise in inorganic mechanisms and fast reaction techniques through work with Geoff Sykes and Manfred Eigen, joined the Unit in 1972.

Cambridge graduate David, with expertise in EPR, was already there and working for Bob Bray. The Unit was custom-built, to include piped nitrogen into the building and huge fermentors for bacterial grow-ups. It was, they tell me, an incredible place to work. The nitrogenase enzyme is, of course, a famously fussy enzyme and extremely sensitive to oxygen, so the piped nitrogen allowed chemistry to be carried out aneorobically. One of the problems is that nitrogenase is never hard up for a substrate – protons or dinitrogen – and so, apparently, it was a very ticklish problem to prevent the enzyme from blowing off (hydrogen) everywhere. Working anaerobically at the bench, they were able to make faster progress and an acetylene reduction assay (from Mike Dilworth) was developed that by-passed the inherent difficulty of assaying for ammonia production.

Thorneley and Lowe

Figure 2: Who is paying? Thorneley (left) and Lowe (right)

Those were the days!
I asked them what I’d been dying to ask for months, about the development of the LT mechanism. The first key experiment was published by Thorneley, Eady and Lowe in 1978 (6). This paper presented kinetic evidence for an enzyme-bound dinitrogen intermediate. Roger and David shared adjacent offices, and in the years that followed the publication of the Nature paper, they ruminated and cogitated on what their observation meant in terms of the mechanism. They were trying to develop a framework for explaining all of the kinetic and spectroscopic data up to that point. This was scientific state-of-the-art….read it and weep! They were using old-fashioned computer punch cards (anyone remember those?) and Roger recalls that they were all stacked high in a deck with knitting needles (provided by David) shoved through to prevent them toppling over. It took six years before their joint deliberations made it into the literature, which was hardly a cracking pace but (they tell me) nobody seemed to mind. They had been assuming that six kinetic steps were needed to understand the complexity of the kinetic data – but in his dreams one night David, apparently, realized that it might be possible to use eight steps instead. So exciting and profound was this revelation that it woke David from his slumber in the middle of the night, and he found himself sat bolt upright in bed. Not quite knowing how to account for this unusual behavior, he proudly declared to his wife his great revelation. “What did your wife say?”, I ask. A pregnant pause. “I can’t remember”, he declares. Nostalgia really is not what it used to be! And I reflect, not for the first time, that the male brain works in strange and surprising ways.

Reaction Mechanism

Figure 3Original hand written Lowe-Thorneley scheme used for simulations with punched card computer input (early 1980s). Note the crossings out. This was created on two A4 sheets of paper, sellotaped together. The complexity of the system is obvious, and is illustrated by the number of partial reactions shown.

The scientific legacy
In the end, their four joint papers covering 32 consecutive pages in one single issue, were all written in long hand and, Roger adds, were published in that format to create “bite-sized chunks” for the community to digest. Any mechanism that can be described in a nutshell more than likely belongs in one, so the reader is referred to Figure 3 and the citations for details. But the LT mechanism is used and referred to regularly (7) as the field moves ahead with exciting new structures, spectroscopy and information on the biosynthesis of the cluster (8,9). And the result of their work – a mechanism which friends and colleagues still use and which still makes a guest appearance on slides all around the world – is a contribution for which bioinorganic chemistry in the UK, and IBDG, should be rightly proud. Those four Biochem. J. papers, and the agonies that surely were part of their conception, have truly stood the test of time. Some papers, as every schoolboy knows, are more equal than others.

David Lowe (former IBDG Chair and Treasurer) and Roger Thorneley (former IBDG Chair) were talking to Emma Raven, at the RSC EPR meeting (Colchester), April 2016

References
1. Lowe, D. J., and Thorneley, R. N. (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady- state kinetics of H2 formation. Biochem J 224, 877-886

2. Thorneley, R. N., and Lowe, D. J. (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady- state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation. Biochem J 224, 887-894

3. Lowe, D. J., and Thorneley, R. N. (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution. Biochem J 224, 895-901

4. Thorneley, R. N., and Lowe, D. J. (1984) The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration. Biochem J 224, 903-909

5. Postgate, J. (1998) The origins of the unit of nitrogen fixation at the University of Sussex. Notes Rec Roy Soc 52, 355- 362. In this interesting historical review, Postgate, who was a microbiologist who had specialised in sulphate reducing bacteria, recalls that Chatt, who was a chemist, considered that Postgate’s experience of “sulphur bugs” made him eminently suitable for the study of nitrogen fixing bateria at UNF, because (Chatt said) nitrogen was only one up diagonally from sulphur in the periodic table.

6. Thorneley, R. N. F., Eady, R. R., and Lowe, D. J. (1978) Biological Nitrogen-Fixation by Way of an Enzyme-Bound Dinitrogen-Hydride Intermediate. Nature 272, 557-558

7. Hoffman, B. M., Lukoyanov, D., Yang, Z. Y., Dean, D. R., and Seefeldt, L. C. (2014) Mechanism of nitrogen fixation by nitrogenase: the next stage. Chemical reviews 114, 4041-4062

8. Ramaswamy, S. (2011) Biochemistry. One atom makes all the difference. Science 334, 914-915

9. Boal, A. K., and Rosenzweig, A. C. (2012) Biochemistry. A radical route for nitrogenase carbide insertion. Science 337, 1617-1618

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