Dalton Discussion No. 8. Metals: centres of biological activity

Inorganic biochemistry presents significant challenges. In particular, investigations of biological systems continue to reveal metal centres that are in an oxidation state and/or environment that has no precedent in conventional coordination chemistry. The power and precision now available in the methodologies of protein identification and expression, structure determination, spectroscopic investigation, and theoretical calculations provide information that allows the relationship between the nature of a metal centre and its biological function to be explored in detail. The programme for the Eighth Dalton Discussion, Metals: Centres of Biological Activity, held in the School of Chemistry of the University of Nottingham on 7–9 September, 2005, was designed with reference to the above perspective and this special issue of Dalton Transactions contains the Keynote and Discussion papers that were presented and discussed at the conference. The pattern of the meeting was similar that of previous Dalton Discussions, in that the presenting authors submitted the manuscript of their Keynote or Discussion paper and was each was refereed, printed, and circulated to delegates prior to the conference. This familiarity with the science meant that, after the Keynote lecture in each session, the presenting authors could summarise the scientific content of their papers in a ten-minute talk, leaving ample time for discussion following each presentation. Also, after lunch on the second day, over thirty young researchers presented posters; each presenter gave a one-minute summary and then discussed the new science accomplished with other delegates on a one-to-one basis by the poster. These poster presentations represented a significant component of the meeting.

The meeting considered a diverse range of biological systems, the function of which require one (or more) metal(s), that are currently under investigation by a comprehensive portfolio of biological, chemical, physical, and theoretical techniques. The interdisciplinary nature of the research, and the commitment and enthusiasm of the delegates ensured lively and thought provoking discussions throughout the meeting. In some cases, the discussion was specific to a particular presentation, but often ranged much wider as common denominators and key themes emerged.

The organising committee would like to thank each of the speakers and participants for making Dalton Discussion 8 such a great scientific success, by fully engaging in the spirit of the meeting. Also, the organising committee is very appreciative of the sterling work performed by members of staff of the RSC Conference Office and Dalton Transactions, the professional expertise and personal involvement of whom ensured that the conference ran smoothly and efficiently throughout. Also, we thank members of the Inorganic Biochemistry Discussion Group, not only for their significant and constructive support during the planning of the meeting, but also for their many and valuable scientific contributions to the proceedings.

The conference comprised of four main sessions, each focussed on key advances in one class of metalloprotein:

 

Haem iron: Nature's most versatile redox centre

Multi-copper enzymes and their chemical analogues

Nickel enzymes: new perspectives and challenges

Molybdenum and tungsten enzymes: nature and function

 

It is difficult and inappropriate to attempt to summarise all of the matters raised in the discussions associated with each of the four half-day sessions in this short Editorial. Therefore, we have attempted to present a distillation of the key points and themes that emerged from each session. We apologise for any omissions and/or inaccuracies in the following account.

Haem iron: Nature's most versatile redox centre

Steve Chapman successfully opened the meeting with his Keynote lecture, in which he reported recent researches on the structural and functional features of multi-haem cytochromes from the Geobacter, Shewanella and Desulfovibrio species of bacteria.¹ Some of these bacteria contain genes for up to seventy different multi-haem proteins, one of which contains an astonishing twenty-seven haem groups. It has become clear that there is a high degree of conservation of the haem arrangements within these proteins, even when there is no clear global structural and sequence analogy between individual proteins. The theme of the discussion following this paper (and of those of other papers later in the meeting) noted that the arrangements of certain redox active prosthetic groups could have evolved, and are continuing to evolve, to maximise the efficiency of electron transfer to and from the active site. This theme was revisited after Stuart Ferguson's presentation that addressed the fundamental issue as to why Nature goes to the trouble of modifying iron protoporphyrin IX, to form c-type and d₁-type cytochromes that achieve specific biological tasks.²

The significant increase in the number and extent of application of modern computational approaches, including DFT and QM/MM methods, to probe the electronic structure and reactivity of metalloenzymes within the frameworks provided by X-ray crystallography was clearly apparent throughout the meeting. Jeremy Harvey³ and Radu Silaghi-Dumitrescu⁴ used these methods as elegant and effective probes of the nature of compound I in cytochrome c peroxidase and ascorbate peroxidase. The discussion that followed these papers focussed on the role of the electrostatic potential associated with the protein around the active site in controlling the electronic structures of key intermediates in the catalytic cycle of these enzymes.

The theme of dioxygen/peroxide activation by haem enzymes continued with Chris Cooper's paper on the reaction intermediates of myoglobin and hydrogen peroxide, in which his research group provided an excellent example of the formation of a true “compound 0” as a primary intermediate⁵ and Harriet Seward's description of some elegant work on two mutants of P450 targeted toward the synthesis of new robust biocatalysts.⁶

Dennis Stuehr moved our attention toward the redox transitions of haem and tetrahydrobiopterin in nitric oxide synthase (NOS).⁷ In this paper he identified key π-stacking interactions between protein residues and tetrahydrobiopterin that can modulate the redox potentials of this prosthetic group. Gary Silkstone followed with a detailed flash photolytic study of the photo-disassociation and subsequent re-association of CO from a chemically modified form of cytochrome c that possesses a vacant sixth coordination site at the haem centre. These studies provide nanosecond insight into the dynamics of this reaction and reveal important differences between the chemistry of the haem centres cytochrome c and myoglobin.⁸ Kate Brown described site-directed mutagenesis studies of three conserved residues located near the haem of catalase peroxidase from Mycobacterium tuberculosis. The subsequent discussion focused on the role of the hydrogen-bonding network around the haem site in maintaining catalytic rather than peroxidatic activity in catalase peroxidase.⁹

Brian Hoffmann's contribution to the meeting¹⁰ described the use of EPR and ¹³C and ¹⁹F ENDOR to examine the geometry of binding of camphor and selected derivatives as substrates of cytochrome P450cam and its T252A mutant. Unfortunately, Brian was not able to attend the meeting to present and discuss the interesting results obtained.

Multi-copper enzymes and their chemical analogues

X-Ray crystallography continues to reveal new and unusual coordination geometries for metal centres in metalloproteins and the work described in Amy Rosenzweig's Keynote lecture certainly followed this trend.¹¹Amy reported a new X-ray crystal structure of the copper-containing membrane-bound methane monooxygenase that has represented one of the major unsolved problems in biological inorganic chemistry for the past twenty years. Almost none of the key structural features of this enzyme had been predicted and, consequently, the solution of this structure represents a significant advance in our understanding in the nature and function of this enzyme. The identification of two new novel copper centres, one mononuclear and the other binuclear and both of which possess an unusual coordination geometry, prompted considerable discussion. A particular point of interest was the significance of the unusually short separation between the copper atoms (ca. 2.6 Å) of the binuclear centre, especially in the context of the of Cu–Cu distances (3.0–3.5 Å) in the chemical analogues of binuclear copper centres in proteins reported by Shinobu Itoh¹² and Martin Feiters.¹³

The theme of unusual multi-copper centres continued with Andrew Thomson's summary of stopped-flow experiments designed to probe the kinetics of the electron transfer steps between cytochrome c and Cu_A in nitrous oxide reductase,¹⁴ and between Cu_A and the unusual Cu_Z cluster in this enzyme. This topic was augmented by Isabel Bento's proposal of a new mechanism for CotA laccase from Bacillus subtilis, based on recent X-ray crystallographic data.¹⁵

Chris Dennison described some elegant site directed mutagenesis studies, designed to investigate the influence of axial ligand interactions with the Type 1 Cu centre, and also the effect of the length and structure of the ligand-containing loop.¹⁶ This paper prompted suggestions of the feasibility of engineering multi-copper sites into the loop regions of the cupredoxins as analogues of these fascinating redox active centres.

Patrick Gamez ended the copper session with a report on the design and activity of new biologically inspired catalysts¹⁷ for copper-mediated oxidative coupling reactions between nucleophiles and 2,4,6-trimethylphenol. This account effectively reinforced the lessons that can be learnt from biology for the design of new large-scale industrially relevant catalysts.

Nickel enzymes: new perspectives and challenges

Fraser Armstrong opened this session with an excellent summary of his group's recent electrochemical experiments on the [NiFe] hydrogenases and showed that this technique is very effective and can map all of the spectroscopically and mechanistically significant states of this enzyme.¹⁸ His presentation ended with the ground-breaking demonstration of the use of laccase- and hydrogenase-coated electrodes in the construction of a membrane-less fuel-cell, as an alternative to cells that employ platinum electrodes. This clearly illustrated both the industrial and environmental relevance of these enzymes. During the subsequent discussion, links were drawn to Steve Chapman's lecture regarding the nature of the prosthetic groups that mediate the electron transport chain to and from a catalytically active site. In particular Fraser noted that the [Fe₃S₄] centre in [NiFe] hydrogenase may be redundant, an observation that leads to the suggestion that the electron-transfer chain in this enzyme may still be evolving.

Juan Fontecilla-Camps described some exciting X-ray crystallographic studies on the peculiar C-cluster active site of the nickel-containing CO dehydrogenase.¹⁹ Whilst this cluster has recently received considerable attention, the mechanism of the catalysis accomplished by this enzyme is not clear. However, Juan's proposed catalytic cycle, derived from the recent crystallographic studies accomplished, provided an excellent framework for the discussion of the nature of the possible intermediates within the catalytic cycle.

Urease was first isolated in a crystallised form over 75 years ago and since then the enzyme has formed the paradigm to illustrate the principles of enzyme structure, mechanism, and catalysis. Nevertheless, the precise details of the mechanism of the catalysis accomplished by urease remain unresolved. Mike Hall attempted to address this deficiency by reporting detailed, high-level density functional calculations on a urease model complex, in which his predicted reaction coordinates effectively rationalises the experimental behaviour of the synthetic system.²⁰

Mechanistic uncertainties also exist for the Ni-containing methyl-coenzyme M reductase enzyme, as Ulrich Ermler demonstrated in his presentation.²¹ He described the current views concerning the mechanism of the reaction between methyl-coenzyme M and coenzyme B, mediated by the unusual Ni-porphinoid F₄₃₀. Also, he suggested that new and surprising discoveries await further researches into the nature of various short-lived intermediates only tentatively identified in the catalytic cycle of this enzyme.

Molybdenum and tungsten enzymes: nature and function

Ralph Mendel presented the Keynote lecture for this session,²² describing the mechanism of Mo uptake into cells, nature of molybdopterin and detailed the various steps involved in its biosynthesis, the insertion of Mo into molybdopterin and the nature of the catalytic centre in each of the three classes of molybdoenzymes. Two potentially important medical advances have emerged from these investigations. Firstly, a link between Cu and Mo metabolism has been demonstrated, since Cu is used to protect the dithiolene group of molybdopterin prior to Mo insertion; this may be significant in respect of the nature of the two diseases, Wilson's and Menkes, that are associated with disorders in the Cu metabolism of humans. Secondly, a deficiency in the availability of molybdenum enzymes is a well-recognised defect of plants and humans. Presently, there is no treatment for this distressing condition that leads to death in early childhood. However, genetic analysis has shown that the majority of the individuals with this condition have defects in the first step of molybdopterin biosynthesis, i.e. the formation of “precursor Z”. Mendel's group have over-produced precursor Z in E. coli and injection of this has successfully restored the normal metabolism of mice genetically engineered to be defective in molybdopterin biosynthesis. Clinical trials of this treatment are about to commence.

With the scene having been successfully set, the remainder of the session started by considering the nature and function of the metal centre, the nature of the catalysis effected, the role of the protein and then how these facets were combined with other redox active groups to produce an active enzyme. The stimulation of coordination chemistry that has resulted from the knowledge available for the Mo and W enzymes was clearly demonstrated by the presentations of Stephen Sproules,²³ Hideki Sugimoto,²⁴ and Ebbe Nordlander.²⁵ Stephen described the synthesis and characterisation of a range of new Mo and W complexes involving pyrazolylborate ligands that have allowed a comparison between true and pseudo-dithiolene complexes in analogous chemical environments. Hideki detailed the synthesis and nature of a new series of Mo(IV), Mo(V), and Mo(VI) 1,2-benzenedithiolate complexes, their redox activity and participation in oxygen atom transfer reactions. Ebbe continued the theme of oxygen atom transfer and described the development of functional model for the active site of the Mo oxotrasferase enzymes.

Ian Hillier has used density functional theory to analyse the contributions to the activation energy for oxygen atom transfer at the catalytic centre of the DMSO reducatses.²⁶ The results of the calculations are informative in respect of comparisons between: (i) Mo and W at the active site; (ii) the pyran ring of molybdopterin open and closed, and (iii) the active site in vacuo and within the protein matrix. The role of the protein in the function of sulfite oxidase was considered in detail by Erkan Karakas, on the basis of information obtained from a series of crystallographic studies.²⁷ This led to a stimulating discussion of the role of particular amino acids in the operation of this enzyme, including electron transfer between the haem and Mo centres. Julia Butt described the value of protein film voltammetry to investigate the function of two nitrate reductases.²⁸ Studies over a range of temperatures has allowed the activation energy for nitrate reduction to be determined. The scientific programme concluded with a discussion of the nature of several of the metalloenzymes considered in the meeting and whether their structure was “the finished, optimised product of evolution” or “work in progress”.

Conclusions

This was a most enjoyable and informative meeting that amply demonstrated the significance of research into metalloproteins, including the metabolism of all life forms, medical treatments and diagnoses, the environment and alternative sources of energy. A truly international cast presented a range of exciting and interdisciplinary research, exchanged ideas freely, and made constructive suggestions for the development of many projects. We were delighted with the presence and participation of so many young scientists. The future of this field, whilst challenging, is bright.

 

Dave Garner

University of Nottingham

 

Jon McMaster

University of Nottingham

 

Emma Raven

University of Leicester

 

Paul Walton

University of York

References

1. C. G. Mowat and S. K. Chapman, Dalton Trans., 2005, 3381.
2. J. W. A. Allen, P. D. Barker, O. Daltrop, J. M. Stevens, E. J. Tomlinson, N. Sinha, Y. Sambongi and S. J. Ferguson, Dalton Trans., 2005, 3410.
3. C. M. Bathelt, A. Mulholland and J. N. Harvey, Dalton Trans., 2005, 3470.
4. R. Silaghi-Dumitrescu and C. E. Cooper, Dalton Trans., 2005, 3477.
5. C. E. Cooper, M. Jurd, P. Nicholls, M. M. Wankasi, D. A. Svistunenko, B. R. Reeder and M. T. Wilson, Dalton Trans., 2005, 3483.
6. H. E. Seward, H. M. Girvan and A. W. Munro, Dalton Trans., 2005, 3419.
7. D. J. Stuehr, C. C. Wei, Z. Wang and R. Hille, Dalton Trans., 2005, 3427.
8. G. Silkstone, A. Jasaitis, M. H. Vos and M. T. Wilson, Dalton Trans., 2005, 3489.
9. N. A. J. Eady, Jesmin, S. Servos, A. E. G. Cass, J. M. Nagy and K. A. Brown, Dalton Trans., 2005, 3495.
10. S. H. Kim, T.-C. Yag, R. Perera, S. Jin, T. A. Bryson, M. Sono, R. Davydov, J. H. Dawson and B. Hoffmann, Dalton Trans., 2005, 3464.
11. R. L. Lieberman and A. C. Rosenzweig, Dalton Trans., 2005, 3390.
12. T. Osako, S. Terada, T. Tosha, S. Nagatomo, H. Furutachi, S. Fujinami, T. Kitagawa, M. Suzuki and S. Itoh, Dalton Trans., 2005, 3514.
13. V. S. I. Sprakel, M. C. Feiters, W. Meyer-Klaucke, M. Klopstra, J. Brinksma, B. L. Feringa, K. D. Karlin and R. J. M. Nolte, Dalton Trans., 2005, 3522.
14. T. Rasmussen, T. Brittain, B. C. Berks, N. J. Watmough and A. J. Thomson, Dalton Trans., 2005, 3501.
15. I. Bento, L. O. Martins, G. G. Lopes, M. A. Carrondo and P. F. Lindley, Dalton Trans., 2005, 3507.
16. C. Dennison, Dalton Trans., 2005, 3436.
17. C. Boldron, Ş. Őzalp-Yaman, P. Gamez, D. M. Tooke, A. L. Spek and J. Reedijk, Dalton Trans., 2005, 3535.
18. K. A. Vincent, J. A. Cracknell, A. Parkin and F. A. Armstrong, Dalton Trans., 2005, 3397.
19. A. Volbeda and J. C. Fontecilla-Camps, Dalton Trans., 2005, 3443.
20. C. Beddie, C. E. Webster and M. B. Hall, Dalton Trans., 2005, 3542.
21. U. Ermler, Dalton Trans., 2005, 3451.
22. R. R. Mendel, Dalton Trans., 2005, 3404.
23. S. A. Sproules, H. T. Morgan, C. J. Doonan, J. M. White and C. G. Young, Dalton Trans., 2005, 3552.
24. H. Sugimoto, M. Tarumizu, K. Tanaka, H. Miyake and H. Tsukube, Dalton Trans., 2005, 3558.
25. A. Thapper, A. Behrens, J. Fryxelius, M. H. Johansson, F. Prestopino, M. Czaun, D. Rehder and E. Nordlander, Dalton Trans., 2005, 3566.
26. J. P. MacNamara, I. H. Hillier, T. S. Bhachu and C. D. Garner, Dalton Trans., 2005, 3572.
27. E. Karakas and C. Kisker, Dalton Trans., 2005, 3459.
28. S. J. Field, N. P. Thornton, L. J. Anderson, A. J. Gates, A. Reilly, B. J. N. Jepson, D. J. Richardson, S. J. George, M. R. Cheesman and J. N. Butt, Dalton Trans., 2005, 3580.

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