The MolybdenumâIron Protein of Nitrogenase - ACS Symposium

Chapter 17

The Molybdenum—Iron Protein of Nitrogenase Structural and Functional Features of Metal Cluster Prosthetic Groups W. H. Orme-Johnson

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Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139

Current proposals for structures of the two recognized types of metal-sulfur clusters in nitrogenase are compared to chemical and spectroscopic evidence in hand. It appears that the P-clusters are pairs of linked Fe S cubes, and the most reasonable interpretation of the oxidation state of the resting enzyme, prior to ATP- coupled injection of electrons from the Fe protein, suggests that the Fe atoms are all ferrous, making it appear that electrons added to such clusters would create a strongly reducing entity in the protein, and further suggesting that the P-clusters donate reducing equivalents to the M (cofactor) clusters, the presumed locale of a dinitrogen reduction. The reported trigonal geometry of six of the Fe atoms in the M centers immediately suggests that these have bound hydride unseen in the crystal structure. Enzyme turned over with ATP and reductant, in D2O, failed in initial experiments to yield EPR evidence for the expected strongly coupled deuterons. If the resting state has a powerful reductant (native P-clusters) and a H reducing site (M -centers) one has to suppose that ATP facilitates the transfer of e between these centers, in addition to the already observed binding of ATP to the Fe protein component of nitrogenase. 4

4

+-

-

Nitrogenase, the enzyme responsible for the ATP-driven reduction of N2 to N H 3 , catalyzes the reaction: N + 16MgATP + 8H+ + 8eq.e" ->2NH + 16MgADP + 16Pi 2

3

This process is evidently energy-prodigal and involves air-sensitive components (as expected, that which will reduce N2 will react readily with O2), as well as being the result of the actions of at least twenty gene

0097-6156/93/0535-0257$06.00/0 © 1993 American Chemical Society

In Molybdenum Enzymes, Cofactors, and Model Systems; Stiefel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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MOLYBDENUM ENZYMES, COFACTORS, AND MODEL SYSTEMS


products (Review: 2). Fortunately for the bioinorganic chemist, nitrogenase is a rather languid catalyst, so that rapidly growing nitrogen-fixing bacterial cells must have 1-5% of their cell protein in the form of this enzyme, somewhat making up for the not inconsiderable lack of joy occasioned by the need for anaerobic Schlenk procedures in essentially every phase of nitrogenase research. The enzyme is composed of two proteins, the Fe Protein (54Kd dimer, containing a single Fe4S4 center) and the MoFe protein (240 Kd#2/ $2, with 30 Fe and 2 Mo atoms) (1) which act in concert. The process is only derepressed in cells starved of a ready source of fixed nitrogen. The striking requirement for MgATP, in a process that is overall already spontaneous, under typical cellular conditions, is of great current interest. The need for ATP obviously derives from the endoergonic nature (2) of the initial step in the reduction of dinitrogen (Figure 1). Early on, we were able to demonstrate (3) that the overall process is as shown in Figure 2. Mechanisms. Thorneley and Lowe, building on an extensive analysis of steady state and pre-steady state kinetic data, subsequently hypothesized (4) that the molecular mechanism of nitrogenase action involves two interlocking cycles (Figure 3). In this model Eo through E7 represent successively reduced states of the MoFe protein, while the triple sets of arrows between the E states depict the action of the Fe protein cycle as theATP-driven electron transfer into the MoFe protein. n

Note that a key feature of this diagram is that NH3 is released prior to the final two electron- transfer cycles, i.e., at least some ATP hydrolysis can have no direct connection to N2 reduction. This hypothesis further directs attention to the concept of the enzyme nitrogenase as catalyzing the functional reverse of oxidative phosphorylation, i.e. using ATP hydrolysis to create intermediate states, which store low-potential reducing equivalents, rather than a role for direct ATP-N2 interaction. Clusters. As referred to below, we made Mossbauer measurements on reversibly oxidized MoFe protein to show that the twenty to thirty iron atoms were disposed into clusters:two M-centers, corresponding (5) to the cofactor originally extracted by Shah and Brill (6) , and four Pclusters (7), which were ultimately deduced from the Mossbauer parameters to be organized into two pairs (8) with each half of the pair differing slightly from the other. Using thiophenol to replace the protein thiols, the P-clusters can be extruded from the MoFe protein as individual Fe4S4 cubanes. According to their behavior during redox titrations, the pairs of clusters are strongly interacting. In contrast, the unperturbed EPR spectrum of the M-centers suggested that they were far enough apart (>10Â) so as to give no indications of magnetic interactions. Recently, we observed an integer spin EPR


17. ORME-JOHNSON

Molybdenum-Iron Protein of Nitrogenase

Energy Profile for N Reduction


2

+ 182kJ

1 ο ο*

-189kJ

Reaction Coordinate

Figure 1 Thermodynamics of dinitrogen reduction. Data from (2). (Courtesy of David Wright). Overol)

Nitroqenose Mechonlsm ferredoxins, other carriers

Energy yielding metabolism

2H

2

H

2

Protein I _ £ j MoFe Protein I

MgADP 4-

Pi

1

/

6H+N

2

\

2NH

3

Figure 2 Overall mechanism of nitrogen fixation. (Reproduced with permission from reference 1. Copyright 1985 Annual Reviews, Inc.)


259

260

MOLYBDENUM ENZYMES, COFACTORS, A N D M O D E L SYSTEMS


signal originating from a MoFe protein sample from which 2e-/Pcluster pair had been removed, thus proving the presence of 2e-/8Fe electron transfer units (9). Structures. In an initial crystallographic analysis, an interpretation of 5Â data taken with two x-ray wavelengths allowed Bolin to suggest the following arrangement (Figure 4) of clusters in the MoFe protein from Clostridium pasteurianum (Cp) (20). Recently, Bolin further proposed that the P-clusters are two Fe4S4 centers joined with a common hexavalent corner S atom, some 19Â from FeMoco and 70Â from the symmetrically disposed pair across the two-fold axis (ACS Meeting, Washington, D.C. August 23-25, 1992); see also Chapter 12 for a different interpretation of the data. Our spectroscopic findings referred to above are entirely consistent with the general crystallographic disposition of the clusters, although it is not apparent that the sulfur bridged P-cluster structure of Bolin would yield Fe4S4 cubes on treatment with thiophenol. Our understanding of the intimate structure of the MoFe protein was dramatically enhanced by an atomic-resolution structure proposed by K i m and Rees (22) for the enzyme from Azotobacter vinelandii (Av). On the basis of sequence homology and spectroscopic parameters, the MoFe proteins of Cp and Av are identical at the level of the cofactor structure and the chemical mechanism of N2 reduction. While the two reported structures agree on the general organization of the M-centers and P-cluster pairs, Kim and Rees propose that the Pclusters are Fe4S4 cubes bridged in pairs by cysteine thiolate ligands coordinating to two Fe atoms. Such a structure is in agreement with the cluster extrusion experiments. They also propose the following structure (Figure 5A) for the FeMo cofactor. Figure 5B shows a structure we hypothesize to be the N2 adduct (cf. an earlier proposal; 22), based on K i m and Rees' crystallographic data). Bolin has also proposed a structure of the cofactor which is similar in many features but has in addition a hexavalent S atom within the Fe cage which converts the three-coordinate peripheral Fe atoms to four coordinate (ACS Meeting, Washington, DC, August 23-25, 1992); more recently (AAAS meeting, Boston, February 13, 1993), Bolin stated that newer interpretations of this data agree exactly with K i m and Rees' work; see also Chapter 12. Previous studies have established that the M-centers yield extractable cofactors when the protein is appropriately unfolded (5,12,13). We have published a highly efficient methodology for such extractions. When the MoFe protein is adsorbed onto DEAE-cellulose and the aqueous buffer is exchanged for DMF or other organic solvents, organic salts (e.g. Et4N Cl ) can be used to disrupt the ionic interactions +

_



ORME-JOHNSON

H

2

k

7

N


Λ

, 2

u

H

2

\ > 1E N H

11

261

4

2

2

pH Ο or > N H 2

2NH, pH Ο *

Ii

pairs. One has wondered for some time (e.g., reference 25 ) wether this pumping might involve direct interaction of ATP, based on the Fe protein, with P-clusters. The recent suggestion, that Pcluster pairs bridge between two protein subunits (11) and that it is plausible that Fe protein docking take place in this neighborhood, makes it of great interest to experimentally test hypotheses about ATP/cluster interactions in nitrogenase during turnovers. Trigonal Fe Atoms i n the Cofactor? The structure proposed by Kim and Rees (Figure 4A) clearly includes planar, three-coordinated Fe atoms at six of the seven positions proposed to be iron sites. One is concerned with the state of bonding that would give rise to such structures; precedent in Fe/S systems (27) clearly favors fourcoordinate, essentially tetrahedral Fe site symmetry in known examples, including ferredoxin centers. The cofactor is intriguingly different in this respect, including having at least five Fe sites distinguishable by ENDOR (28). There are two explanations that immediately occur to one: first, that the cofactor is an electron rich, delocalized system and that in the resting state of the enzyme a kind of Faraday cage effect has apparently favored the observed hybridzation scheme. Second, the cofactor is really a kind of metal hydride reservoir and therefore the missing Fe ligand is a hydrogen atom in each case. This would not be easily visible in x-ray analyses of proteins, but it certainly would be seen by ENDOR or ESEEM of protein soaked in D2O under turnover (electron transfer) conditions. Protein passively exposed to D2O reveals a series of exchangeable protons by these means (29). We have recently done the turnover experiment; in the event, no evidence for electron transfer dependant exchange has been seen up to now (R. Pollock, B. Hoffman, W.H. Orme-Johnson, in progress). Completion of a high resolution structure of isolated cofactor would address this point more effectively. In the meantime, it is useful to note that as a hydrogen evolving system, there must be electron transfer dependent interaction of the MoFe protein with solvent protons. Since the resting state, with the useful S=3/2 cofactor signal,


268



does not evolve H2, the lack of evidence for ATP-dependant exchange is disappointing but not surprising. The resting state presumably has no unspent hydride equivalents, according to these preliminary results. Which leaves one exactly where one started: with trigonal Fe atoms, which still invite speculation without at this point producing much certainty as to their significance, and, so far, also leave untouched the question, where do the hydride equivalents reside, in H2" evolving, N2" reducing states of nitrogenase? The smart money would bet on the M-centers, but decisive work remains to be done. Acknowledgements: This work was supported by the N I H under GM28358. The author thanks his coworkers, particularly Maria G. Mitchell, David Wright, Patricia Christie, Normand Cloutier, Jacquin Niles, Robert Pollock and Jeremy Selengut, for their timely help, as well as acknowledging his extended and profitable collaborations with Eckard Munck and Brian Hoffman and their coworkers, and the work of previous pre- and postdoctoral students, cited in the references and figure legends. The insights and criticisms of Fred Ausubel, Alison Burgess, Jay Hickman, Anne-Francis Miller and Dick Schrock remain valuable resources in the areas of genetics, metal metabolism, and inorganic mechanisms, respectively. Literature Cited 1. Orme-Johnson, W . H . Molecular Basis of Biological Nitrogen Fixation, Ann. Rev. Biophys. Chem., 1985,14, 419-459. 2. Subcommittee on Ammonia, National Research Council, "Ammonia/' University Park Press, 1979 . Baltimore, MD. 3. Orme-Johnson, W.H., Hamilton, W.D., Jones, T.L., Tso, M-Y W., Burris, R . H . , Shah, V.K., and Brill, W.J. Electron Paramagnetic Resonance of Nitrogenase and Nitrogenase Components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP, Proc. Natl. Acad. Sci., 1972,69,3142-3145. 4. Lowe, D.J., Thorneley, R.N.F. The Mechanism of Klebsiella pneumoniae Nitrogenase Action. Biochem J. 1984., 224, 877-886. 5. Rawlings, J., Shah, V.K. Chisnell, J.R., Brill, W.J., Zimmermann, R. Munck, E., Orme-Johnson, W.H. Novel Metal Cluster in the IronMolybdenum Cofactor of Nitrogenase, J.Biol Chem., 1978, 253, 10011004. 6. Shah, V.K., Brill, W.J. Isolation of an Iron-molybdenum Cofactor from Nitrogenase, Proc. Natl. Acad. Sci. U.S.A., 1977, 74, 3249-3256. 7. Huynh, B.W., Munck, E., Orme-Johnson, W.H. Nitrogenase XI: Mossbauer Studies on the Cofactor Centers of the MoFe Protein from Azotobacter vinelandii OP, Biochim. Biophys. Acta, 1979,527,192-203. 8. McLean, P.A., Vasilios, P., Orme-Johnson, W . H . , Munck, E. Isotopic Hybrids of Nitrogenase Mossbauer Study of MoFe protein with selective ^ Fe-Enrichment of the P-Cluster. /. Biol. Chem., 1988,262, 12902-12903. 7


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ORME-JOHNSON


9. Surerus, K.K., Hendrich, M.P. Christie, P.D., Rottgardt, D., OrmeJohnson, W.H., Munck, E. Mossbauer and Integer-Spin EPR of the Oxidized P-Clusters of Nitrogenase: P ° is a Non-Kramers System with a Nearly Degenerate Ground Doublet. /. Am. Chem. Soc, 1992, 114, No. 228579-8590. 10. Bolin, J.T. Ronco, L . E . , Mortenson, L . E . , Morgan, T.W., Williamson, M . N.-h. Xuong. Structures of the Nitrogenase MoFe Protein: Spatial Distribution of the Intrinsic Metal Atoms Determined by X-ray Anomalous Scattering. Nitrogen Fixation Proc. 8th Int. Congress. Knoxville, Tennessee, Peter M . Gresshoff, L. Evans, Roth, Gary Stacey, William E. Newton, (eds). Chapman and Hall, New York, 1990,117-124. 11. Kim, J . , Rees, D.C. Structural Models for the Metal Centers in the Nitrogenase Molybdenum-Iron Protein. Science, 1992,257,1677-1682. 12. Orme-Johnson, W.H. Perspectives: Nitrogenase Structure: Where to Now? Science, 1992,257,1639-40. 13. McLean, P.A., Wink, D . A . , Chapman, S.K., Hickman, A . B . , McKillop, D.M., Orme-Johnson, W.H. A New Method for Extraction of Iron-Molybdenum Cofactor (FEMOCO) from Nitrogenase Adsorbed to DEAE Cellulose: I. Effects of Anions, Cations, and Preextraction Treatments. Biochemistry, 1989,28, 9402-9406 14. Wink, D.A., McLean, P.A., Hickman, A.B., Orme-Johnson, W . H . A N e w Method for Extraction of Iron-Molybdenum Cofactor (FEMOCO), from Nitrogenase adsorbed on DEAE Cellulose: II. Solubilization of the Iron-Molybdenum Cofactor (FEMOCO) in a Wide Range of Organic Solvents. Biochemistry, 1989,28, 9407-9412. 15. Burgess, B. The Iron-Molybdenum Cofactor of Nitrogenase. Chem. Rev,. 1990, 90,1377-1406. 16. Nelson, M.J., Levy, M.A., Orme-Johnson, W.H. Metal and Sulfur Composition of Iron-Molybdenum Cofactor of Nitrogenase, Proc. Natl Acad. Sci., 1983, 80,147-150. 17. Hoover, T.R., Imperial, J . , Ludden, P.W., Shah, V.K. Homocitrate Is a Component of the Iron-Molybdenum Cofactor of Nitrogenase. Biochem., 1989,28, 2768-2771. 18. Conradson, S.D., Burgess, B.K., Vaughn, S.A. , R œ , A.L., Hedman, Britt, Hodgson, K.O., Holm, R.H. Cyanide and Methylisocyannide Binding to the Isolated Iron-Molybdenum Cofactor of Nitrogenase. /. Biol. Chem., 1989,264,15967-15974. 19. Georgiadis, M . M . , Komiyz, H., Chakrabarti, P., Woo, D., Kornuc, J. H . , Rees, D.C. Crystallographic Structure of the Nitrogenase Iron Protein from Azotobacter vinelandii, Science. 1992,257,1653-1656. 20. Lindahl, P.A., Day, E.P., Kent, T.A., Orme-Johnson, W.H., Munck, E. Mossbauer, EPR and Magnetic Susceptibility of the Iron Protein from Azotobacter vinelandii Nitrogenase. /. Biol. Chem. ,1985,260,11160-73. 21. Pretorious, I.M., Rawlings, D.E., O'Neill, E.G., Jones, W.A., Kirby, R., Woods, D.R. Nucleotide Sequence of the Gene Encoding the Nitrogenase Iron Protein of Thiobacillus ferrooxidans. J. Bact., 1987,169, 367-370. x


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22. Lindahl, P.A., Gorelick, N.J., Munck, E., Orme-Johnson, W.H. EPR and Mossbauer Studies of Nucleotide-bound Nitrogenase Iron Protein from Azotobacter vinelandii J. Biol Chem., 1987,262,14945-14953. 23. Thornley, R.N.F. Kinetics and Mechanisms of ATP Hydrolysis, Electron Transfers and Proton Release by Klebsiella pneumoniae Nitrogenase. Nitrogen Fixation, Proc. 8th Int. Congress. Knoxville, Tennessee. Peter M . Gresshoff, L. Evans Roth, Gary Stacey, William E. Newton, eds. Chapman and Hall, New York, 1990,103-109. 24. Lowery, R.G., Chang, C.L., Davis, L.C., McKenna, M - C , Stephens, P.J., Ludden, P.W. Substitution of Histidine for Arginine-101 of Dinitrogenase Reductase Disrupts Electron Transfer to Dinitrogenase. Biochem., 1989,28,1206-1212. 25. Orme-Johnson, W.H., Orme-Johnson, N.R., Touton, C , Emptage, M . , Henzl, M . , Rawlings, J., Jacobson, K., Smith, J.P., Mims, W.B., Huynh, B.H., Munck, E., Jacob, G.S. Spectroscopic and Chemical Evidence for the Nature and Role of Metal Centers in Nitrogenase and Nitrate Reductase in Molybdenum Chemistry of Biological Significance, W.E. Newton, S. Otsuka, eds., (International Symposium, held at Lake Biwa, Japan, April 10-13). Plenum, New York City, 1979,85-94; 26. Zimmermann, R., Munck, E., Brill, W.J., Shah, V.K., Henzl, M.T., Rawlings, J., Orme-Johnson. W.H. Nitrogenase X: Mossbauer and EPR Studies on Reversibly Oxized MoFe Protein from Azotobacter vinelandii OP, Biochim. Biophys. Acta,, 1978,537,185-207. 27. Averill, B.A., Orme-Johnson, W . H . Iron-Sulfur Proteins and Their Synthetic Analogs, in Metal Ions in Biological Systems, 7, Ch. 4, Helmut Sigel, ed., Marcel Dekker, Inc. Basel, Switzerland, 1978, 127-183. 28. True, A.E., Nelson, M.J., Venters, R.A., Orme-Johnson, W . H . , Hoffman, B.M. Determination of the F e Hyperfine Tensors for Five Distinct Iron Sites of the Iron-Molybdenum Cofactor Within the Molybdenum-Iron Protein of Azotobacter vinelandii, J. Am. Chem. Soc, 1988,110,1935-1943. 29. True, A.E., McLean, Nelson, M.J., Orme-Johnson, W.H., Hoffman, B.M. Comparison of Wild-Type and nifV Mutant Molybdenum-Iron Proteins of Nitrogenase from Klebsiella pneumoniae by E N D O R Spectroscopy. /. Am. Chem. Soc 1990,112, 651-657. 30. Watt, G.D., Burns, A . Tennent, D.L. Stoichiometry and Spectral Properties of the MoFe Cofactor and Noncofactor Redox Centers in the MoFe Protein of Nitrogenase from Azotobacter vinelandii. Biochemistry 1981,20,7272-7277. 57

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