Metal Clusters in Proteins - ACS Publications - American Chemical

Chapter 18

Nitrogenase

Downloaded by UNIV OF LIVERPOOL on May 21, 2017 | http://pubs.acs.org Publication Date: June 21, 1988 | doi: 10.1021/bk-1988-0372.ch018

Overview and Recent Results E. I. Stiefel, H. Thomann, H. Jin, R. E. Bare, T. V. Morgan, S. J. N . Burgmayer, and C. L. Coyle Exxon Research and Engineering Company, Route 22 East, Annandale, NJ 08801 The b i o l o g i c a l nitrogen f i x a t i o n process i s introduced. Discussion focusses on the 'Dominant Hypothesis' of nitrogenase composition and functioning. The enzyme system catalyzes the six-electron reduction of N2 to 2 NH3 concomitant with the evolution of H2. ATP hydrolysis drives the process. The two protein components of the enzyme, [Fe] and [FeMo], contain t r a n s i t i o n metal s u l f i d e clusters. Recently, an a l t e r n a t i v e nitrogenase containing vanadium has also been reported. [FeMo] contains the unique FeMo-co s i t e , which has been studied using microbiological, molecular genetic, biochemical, biophysical, chemical and synthetic modelling approaches. Alternative substrates for nitrogenase include a number of unsaturated small molecules. The inorganic chemical l i t e r a t u r e y i e l d s clues for the a c t i v a t i o n of relevant small molecules such as acetylenes. Substrate reactions of nitrogenase also implicate hydrogen a c t i v a t i o n as a key feature of nitrogenase turnover. The d i f f e r e n t ways i n which hydrogen can interact with t r a n s i t i o n metal s u l f i d e c l u s t e r s are discussed. The need for application of sophisticated probes to d i s t i n g u i s h s t r u c t u r a l and mechanistic p o s s i b i l i t i e s i s emphasized. Recent work i s presented on the use of Electron Spin Echo Spectroscopy to probe the r e l a t i o n of the extracted cofactor to the center i n the intact FeMo protein. The process of nitrogen f i x a t i o n i s an e s s e n t i a l part of the nitrogen cycle on the planet e a r t h ( l ) . I t i s estimated that greater than 60% of the N2 that i s ultimately converted to N H 4 i s done so by the nitrogenase enzyme system. The a v a i l a b i l i t y of fixed nitrogen i s often the l i m i t i n g factor i n plant growth(2). To +

0

0097-6156/88/0372-0372$06.00/0 1988 American Chemical Society

Que; Metal Clusters in Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


18.

STIEFEL ET AL.

Nitrogenase: Overview and Recent Results

373

compensate f o r the i n a b i l i t y of natural systems to provide enough nitrogen, man has developed processes to " f i x nitrogen" chemically. By f a r the major process i n use today i s the Haber-Bosch process i n which N2 and H2 are reacted at temperatures between 300-500° C and pressures over 300 atm using (usually) Fe-based c a t a l y s t s Q ) . L i t e r a l l y hundreds of massive chemical plants are used, and these often produce 1,000 tons of NH3/day. In contrast, i n the b i o l o g i c a l process, N2 i s reduced l o c a l l y as needed at room temperature and -0.8 atm pressure. S u p e r f i c i a l l y , i t might seem that the b i o l o g i c a l process i s inherently simpler than the i n d u s t r i a l one. That t h i s i s not the case i s seen f i r s t by the genetic complexity of the b i o l o g i c a l process. Figure 1 shows that at least seventeen genes are required for e f f e c t i v e nitrogen f i x a t i o n i n the bacterium K l e b s i e l l a pneumoniae(4.5). These nif genes specify proteins that are involved i n regulation (nifA and L), pyruvate o x i d a t i o n / f l a v i n reduction (nifJ), electron transfer (nifF for flavodoxin), the subunits of the s t r u c t u r a l proteins of the nitrogenase (nifH, D, K), Fe-S c l u s t e r assembly (nifM) and the biosynthesis of the i r o n molybdenum cofactor, FeMo-co (nifN, B, E, Q, V, H)(5a). I t i s the l a s t two functions, involving the placement of unusual t r a n s i t i o n metal s u l f i d e clusters into the nitrogenase proteins, that cause nitrogenase and i t s components to be appropriately included i n t h i s symposium. In this chapter we f i r s t present what has been called(6) the 'Dominant Hypothesis' f o r the structure and function of molybdenum-based nitrogenases. We summarize the h i s t o r y of the newly discovered a l t e r n a t i v e vanadium-based nitrogenase. The properties of the vanadium-based enzyme point to the importance of the study of alternative substrate reactions i n probing nitrogenase r e a c t i v i t y . These reactions are summarized and used as a s t a r t i n g point to discuss r e s u l t s from inorganic chemistry that are p o t e n t i a l l y relevant to understanding the enzyme. The p o s s i b i l i t i e s for substrate and hydrogen a c t i v a t i o n revealed i n inorganic studies point poignantly to the need for further s t r u c t u r a l , spectroscopic and mechanistic d e f i n i t i o n of the nitrogenase active s i t e s themselves. Such d e f i n i t i o n i s progressing on many fronts and we b r i e f l y describe some of our own e f f o r t s using Electron Spin Echo (ESE) Spectroscopy. The Dominant Hypothesis f o r Molybdenum Nitrogenase(6.7.8.9) The action of the nitrogenase enzyme requires the presence of two component proteins. The larger of the two proteins, sometimes incorrectly(10) designated(ll) as dinitrogenase, i s usually c a l l e d the MoFe or FeMo protein ([MoFe] or [FeMo]). The smaller, c a l l e d the Fe protein or [Fe] i s sometimes incorrectly(10) referred to (II) as dinitrogenase reductase. Figure 2 shows a schematic diagram that summarizes the protein composition and functioning. The Fe protein contains two i d e n t i c a l subunits of M.W. 30,000 which are products of the n i f H gene(12). A single Fe4S4 center i s present i n the protein and appears to be bound between the subunits.(12a) The Fe protein i s reducible by c e l l u l a r reductants such as flavodoxin or ferredoxin or a r t i f i c i a l =


374

METAL CLUSTERS IN PROTEINS 49

6 0 5 0 2 0 2 8 4 2 4 5 2 5 18 5 0 4 0 2 4 6 0 6 0 3 5 120

h i s Q B A L F M

V S U X N E V K D H J


REGULATION

Flavodoxin PyruvateFe protein Flavoproteln Oxidoreductase

FeMo protein

Figure 1 . Nif genes required for nitrogen f i x a t i o n as arranged i n K l e b s i e l l a pneumoniae. The numbers i n the top row are molecular weights i n kilodaltons of the protein gene products o f the respective nif genes whose l e t t e r s are shown below them.

Fe P r o t e i n (65,00a)

F e

S

= 4 4

MoFe P r o t e i n (230,000) e

= P-Cluster [Fe S l 4

Figure 2.

4

FeMoco [MoFe S ] 7

Q

Schematic diagram of the nitrogen f i x i n g enzyme.



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375

reductants such as d i t h i o n i t e or viologens. Its Fe4S4 center undergoes a single one-electron redox process wherein the reduced form i s EPR active and the oxidized form i s diamagnetic. Until quite recently a major mystery involved the low EPR spin-quantitation of the Fe4S4 center compared to the a n a l y t i c a l l y derived values for the number of such centers(6.13). The explanation for the discrepancy now seems to be i n hand(14-17) as i t has been c l e a r l y established that the Fe4S4 center exists i n two spin states, S - 1/2 and S - 3/2. Only the former with i t s g values near 2 was considered i n the e a r l i e r spin quantitations. When the S - 3/2 center with g between 4 and 6 i s also taken into account, the spin-integration c l e a r l y shows one paramagnetic s i t e for each Fe4S4 unit. Model systems(18) and t h e o r e t i c a l studies(19) strongly support the notion that various spin states are possible for Fe4S4 cluster systems. During enzyme turnover t h i s center transfers electrons to the FeMo protein i n single electron steps. The Fe protein binds two molecules of MgATP(20). In the active enzyme system, a minimum of two molecules of MgATP are hydrolyzed to MgADP and phosphate i n conjunction with the transfer of a single electron to the FeMo protein(21). The ATP/2e" r a t i o i s generally accepted to have a minimum value of 4 with higher numbers representing decreased e f f i c i e n c y , possibly due to " f u t i l e c y c l i n g " where back electron transfer from [FeMo] to [Fe] raises the e f f e c t i v e ratio(13,21). Except for an unconfirmed report of reduction by thermallized electrons produced by pulse radiolysis(22), no evidence exists that the FeMo p r o t e i n can be reduced to a c a t a l y t i c a l l y capable form without the Fe protein present. This s i t u a t i o n c a l l s to mind the nomenclatural proposal(11) of Hageman and Burris that [FeMo] be designated as dinitrogenase and [Fe] as dinitrogenase reductase. The incorrectness or, at best, prematurity(10) of this suggestion l i e s i n the i n a b i l i t y of either protein to function c a t a l y t i c a l l y i n the absence of the other. [FeMo] w i l l not reduce N2 or C2H2 i n the absence of [Fe]. [Fe] w i l l not hydrolyze ATP i n the absence of [FeMo]. The nitrogen f i x a t i o n process requires the presence of both proteins. Although mechanistic considerations(23) point to [FeMo] as the substrate binding and reducing protein and [Fe] as the ATP binding locus, c a t a l y t i c reactions have never been consummated by one protein i n the absence of the other (but vide i n f r a f o r the uptake of H2). Therefore, neither p r o t e i n can be considered as an enzyme, which the proposed nomenclature implies. We therefore use the [FeMo] and [Fe] designations i n accord with most workers i n the area. This caveat notwithstanding, the Dominant Hypothesis(6) designates [FeMo] as the protein responsible f o r substrate reduction. The FeMo protein contains an ct2Pl subunit structure due to expression of the nifD and nifK genes(24.25). I t s o v e r a l l M.W. of about 230,000 r e f l e c t s the 50-60,000 M.W. of each of i t s four subunits. The nonprotein composition of 30 Fe, 2 Mo, and 30 s2- betokens the presence of t r a n s i t i o n metal s u l f i d e c l u s t e r s , which are presumed to be the active centers of the protein. Without intending to make any spacial implications, figure 2 shows



376

METAL CLUSTERS IN PROTEINS

the d i s t r i b u t i o n of major clusters according to the Dominant Hypothesis. Four Fe4S4-like clusters appear to be present i n [FeMo] and these have been designated as P-clusters(25). The P c l u s t e r s are, however, by no means ordinary Fe4S4 c l u s t e r s , i f indeed they are Fe4S4 clusters at a l l . P clusters are conspicuous i n UV-VIS and especially, MCD and Mossbauer spectra(6,13). The observed spectra are c l e a r l y not conventional. The clusters have a very high spin i n t h e i r oxidized forms, probably S - 7/2 from recent(28a) EPR studies, and have decidedly inequivalent Fe populations(28). This implies that the putative Fe4S4 clusters are highly distorted, presumably by unsymmetrical coordination from the protein. Moreover, the P clusters do not appear to behave i d e n t i c a l l y to each other under many circumstances. There i s open disagreement as to the redox behavior of t h i s set(6,26,22). Furthermore, an additional Mossbauer signal c a l l e d S may i n fact be part of the P-cluster signal(28). Clearly, the spectroscopic properties of the P c l u s t e r s i n the proteins do not reveal t h e i r s t r u c t u r a l nature. However, extrusion of these clusters from the protein leads to the clear i d e n t i f i c a t i o n of 3-4 Fe4S4 clusters(13,29). Despite the uncertainties inherent i n the extrusion procedure (due to possible cluster rearrangement) the extrusion r e s u l t supports the Dominant Hypothesis, which designates the P centers as Fe4S4 units, a l b e i t highly unusual ones. The P clusters are thought to be involved i n electron transfer and storage presumably providing a reservoir of low p o t e n t i a l electrons to be used by the M center (FeMo-co) i n substrate reduction. The FeMo-co or M center of the FeMo protein has been i d e n t i f i e d spectroscopically(6,13,30) within the protein and has been extracted from the protein into N-methyl formamide(31) and other organic solvents(32.33). Its biochemical authenticity can be assayed by i t s a b i l i t y to activate FeMo protein from a mutant organism that produces protein that lacks the M center(31). The extracted cofactor resembles the M-center unit spectroscopically and s t r u c t u r a l l y as shown i n Table I. I t seems reasonable to presume that the differences are due to v a r i a t i o n i n the l i g a t i o n of the center between the protein and the organic solvent(34)• In the Dominant Hypothesis the FeMo-co center i s presumed to be the substrate binding and reducing s i t e . Strong evidence to support t h i s idea comes from the study of nifW mutants(35-37). (Note that nifV has nothing to do with vanadium). NifV mutants have altered substrate s p e c i f i c i t y insofar as they do not f i x nitrogen i n vivo. In v i t r o H2 evolution by i s o l a t e d n i f V nitrogenase i s i n h i b i t e d by CO unlike the wild type where H2 evolution i s insensitive to CO. Most s i g n i f i c a n t l y , t h i s behavior i s transferred with the FeMo-co unit that i s extracted from the n i f V protein and used to reactivate the FeMo-co-deficient mutant. The reconstituted FeMo protein has CO sensitive H2 evolution(37). The FeMo-co s i t e i s thus c l e a r l y implicated as a major p a r t i c i p a n t i n the substrate reactions of the nitrogenase enzyme complex. The two-component enzyme catalyzes the reduction of N2 to 2NH4+ or of C2H2 to (exclusively) C2H4 and the evolution of H2 with electrons supplied by the reductants named above. ATP


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377

Table I Comparison of the FeMo Protein and Isolated FeMo-co

FeMo Protein (M Center)

a

FeMo-co ( i n NMF)

EPR 4.3

4.8

3.7

3.3

2.01

2.0

Mo-S

2.37(4.5)b,d

2.37(3.1) >

Mo-Fe

2.68(3.5)b,d

2.70(2.6)c,d

Mo-0 or N

2.12(1.7)b,d

2.10(3.1) »


g values

a

EXAFS

c

c

Fe-S

2.25(3.4)e

Fe-Fe

2.66(2.3)®

Fe-Mo

2.76(0.4)©

Fe-0 or N

1.81(1.2)®

d

d

XANES M0O3S3

fits best

f

a

Distance i n A with number of atoms i n parentheses.

b

E a r l i e r study by Cramer, S. P.; Hodgson, K. 0.; Gillam, W. 0.; and Mortenson, L. E. ; J. Am. Chem. S o c , 1978 100, 2748.

c

E a r l i e r study by Burgess, B. K.; Yang, S.-S.; You, C.-B.; L i , J.-G.; Friesen, G. Pan, W.-V.; S t i e f e l , E. I.; Newton, W. E.; Conradson S. D.; Hodgson, K. 0. i n ref. 8, p 71.

dData from Conradson, S. D.; Burgess, B. K.; Newton, W. E.; Mortenson, L. E.; and Hodgson, K. 0. J. Am. Chem. Soc. 1987 109, 7507. e

Antonio, M. R.; Teo, B.-K.; Orme-Johnson, W. H.; Nelson, M. J . ; Groh, S. E.; Lundahl, P. A.; Kauzlarich S. M.; A v e r i l l , B. A. J . Am. Chem. Soc, 1982 104, 4703.

f

Conradson, S. D.; Burgess, B. K.; Newton, W. E.; Hodgson, K. 0.; McDonald, J . W.; McDonald, J . W.; Rubinson, J . F.; Gheller, W. F. ; Mortenson, L. E.; Adams, M. W. W. ; Mascharak, P. K. ; Armstrong W. A.; Holm, R. H. J . An. Chem. S o c , 1985, 107, 7935.


378


hydrolysis occurs concomitantly with electron transfer from [Fe] to [FeMo]. Dissociation of [Fe] from [FeMo] a f t e r this electron transfer i s believed to be the r a t e - l i m i t i n g step i n the o v e r a l l turnover of the enzyme(25).


Vanadium and

Nitrogenase

The association of molybdenum with nitrogen f i x a t i o n was f i r s t reported by Bortels i n 1930(38). This seminal finding opened the door to the characterization of the molybdenum nitrogenases discussed i n the preceding section. The Bortels work has been c i t e d many times and i s often referred to without c i t a t i o n . In addition to nitrogenase, many other Mo-containing enzymes were subsequently sought and found(39). Subsequent to the c l a s s i c 1930 paper, Bortels reported i n 1936(40) that vanadium stimulated nitrogen f i x a t i o n . The 1936 paper was hardly ever c i t e d p r i o r to 1986. Despite a few p o s i t i v e reinforcements, i t was not u n t i l the 1970's that serious attempts were made to i s o l a t e a vanadium nitrogenase. In 1971, two groups reported the successful i s o l a t i o n of a vanadium-containing nitrogenase from Azotobacter vinelandii(41.42). The enzyme was s i m i l a r to the Mo enzyme but was reported to have low a c t i v i t y and an altered substrate s p e c i f i c i t y . Driven i n part by skepticism from the nitrogenase research community, one of the groups c a r e f u l l y reinvestigated t h e i r preparation and found small amounts of molybdenum presumed to be s u f f i c i e n t to account f o r the activity. (The s e l e c t i v i t y difference was not addressed.)(43) The vanadium was thought to play a s t a b i l i z i n g role f o r the FeMo protein, allowing the small amount or active [FeMo] to be e f f e c t i v e l y isolated. Interestingly, the vanadium was said to "substitute" for Mo i n the FeMo nitrogenase. Apparently, no thought was given to the p o s s i b i l i t y of a t r u l y alternative nitrogenase system whose protein as well as metal content d i f f e r e d from that of the Mo nitrogenase. The irreplaceable e s s e n t i a l i t y of molybdenum i n nitrogenase went unchallenged u n t i l 1980 when Bishop and coworkers demonstrated(44) that an alternative nitrogen f i x a t i o n pathway became active i n Azotobacter v i n e l a n d i i when this organism was starved for molybdenum(45). Despite continued skepticism, Bishop persevered and eventually showed that even i n a mutant from which the nifH, D and K genes had been deleted, the alternative system could be e l i c i t e d upon Mo starvation. The c i r c l e was f i n a l l y closed i n 1986 when two groups(46-51) i s o l a t e d the component proteins of the alternative nitrogenase from d i f f e r e n t species of Azotobacter. They showed unequivocally that one component contained vanadium and that neither component contained molybdenum. The p u r i f i c a t i o n and characterization of the V nitrogenase proteins has shown that one component i s extremely s i m i l a r to the Fe protein of nitrogenase. This evidence comes both from the i s o l a t i o n of the protein from Azotobacter vinelandii(46) and from the i d e n t i f i c a t i o n of genetic homology between n i f H (the genetic determinant f o r the subunit of the Fe protein i n the Mo nitrogenase system) and n i f H * (the corresponding



18. STIEFEL ET AL.


379

gene i n the V based system). Both Fe proteins contain an c*2 subunit structure and a single F e 4 S 4 c l u s t e r , which i s EPR active i n i t s reduced state. The FeV protein from Azotobacter v i n e l a n d i i and Azotobacter chroococcum has an ©2^2 subunit structure with metal analysis and spectroscopic properties shown i n Table II i n comparison with properties of [FeMo]. Clearly, the major difference i s the presence of V instead of Mo i n the FeV protein. However, i t i s c l e a r that the two nitrogenase systems are r e a l l y quite s i m i l a r . In each case, two highly 02-sensitive proteins carry out an ATP-dependent reduction of N2 and concomitant evolution of H2. The Fe proteins have the same (a2) subunit structure, c l u s t e r content and spectroscopic signature. The V versions of the larger protein, while somewhat lower i n M.W., have the same subunit structure as t h e i r Mo analogs («2^2) d appear by MCD spectroscopy to contain P-like clusters(48)• The center d i f f e r s from the FeMo system by the s u b s t i t u t i o n of V for Mo. However, the FeV s i t e s t i l l may be an S - 3/2 center (by EPR, although this d i f f e r s from that of the FeMo center) (52) and seems to have V-S and V-Fe distances (by EXAFS)(53,54) s i m i l a r to those i n thiocubane V F e 3 S 4 c l u s t e r s . This observation r e c a l l s the finding that FeMo-co has Mo-S and Mo-Fe distances s i m i l a r to M o F e 3 S 4 thiocubanes. Likewise XANES(53) implicates V S 3 O 3 type coordination i n the V nitrogenase as i t does M 0 S 3 O 3 coordination i n FeMo-co. F i n a l l y , the "FeV cofactor" i s extractable into NMF and can reconstitute the n i f B " FeMo-co-less mutant of the Mo system(55). Clearly, despite the s u b s t i t u t i o n of V for Mo, the properties of the proteins, including t h e i r respective M-Fe-S s i t e s do not appear to d i f f e r d r a s t i c a l l y . However, the compositional changes do have a s i g n i f i c a n t c o r r e l a t i o n i n a l t e r e d reactivity. A major difference between the V and Mo enzymes l i e s i n substrate s p e c i f i c i t y and product formation(51). As c l e a r l y shown i n Table I I , the FeV nitrogenase has a much lower r e a c t i v i t y toward acetylene than does the Mo system. Furthermore, while the FeMo system exclusively produces ethylene from acetylene the FeV system y i e l d s s i g n i f i c a n t amounts of the four-electron reduction product, ethane(51). The detection of ethane i n the acetylene assay may prove a powerful technique for detecting the presence of the V nitrogenase i n natural systems. Moreover, t h i s r e a c t i v i t y pattern i s found i n the n i f B " mutant reconstituted with FeV-co(55). The r e a c t i v i t y change upon going from Mo to V i n otherwise s i m i l a r protein systems c l e a r l y adds weight to the d i r e c t implication of the M-Fe-S center (M - V or Mo) i n substrate reduction. a n

F e V

Substrate

Reactions

In addition to the physiological reaction of N2 reduction, nitrogenase catalyzes a wide v a r i e t y of reactions involving small unsaturated molecules(56). Table III l i s t s key reactants and products for FeMo nitrogenases. A l l substrate reductions involve minimally the transfer of two electrons. Multielectron substrate reductions may involve the accretion of such two-electron


380


Table II Comparison of Nitrogenase

4

Property

Avl ?

240,000


Molecular

Proteins

4

Avl* ?

200,000

a

Acl*50

210,000

weight

C H

N3-

+

2e-

+

3H+

-

>

NH + N

2

N0

+

2e"

+

2H+

-

>

H0 + N

2

>

CH = CH - CH3

C H 2

2

2

/

\

HC

+

2e" + 2H+

2

2

4

3

2

2

CH

Four-Electron Reductions

HCN

+

4e" +

RNC + 4e" + Six-Electron Reductions

4H+

> CH3NH

4H+

>

2

RNHCH3

2

+

6e-

+

6H+

-

>

2NH3

HCN

+

6e"

+

6H+

-

>

CH4 + NH3

HN3

+

6e"

+

6H+

-

>

NH3 + N H4

RNC

+

6e"

+

6H+

-

>

RNH + CH4

RCN

+

6e-

+

6H+

-

>

RCH3 + NH3

N

2

2

Multielectron Reductions

RNC -+ (C H6, C3H6, C3H8) + RNH 2

2


382


processes. Further, except f o r the reduction of N 3 " to NH3 and N ,(57) an equal number of protons and electrons i s transferred to the substrate. In the absence of substrate, two electrons combine with two protons to form H 2 . The active s i t e of nitrogenase seems capable of d e l i v e r i n g the elementary p a r t i c l e s of H2 to the substrate. Of the a l t e r n a t i v e substrates of nitrogenase, many contain t r i p l e bonds i n at least one of t h e i r resonance forms. As discussed i n the next section, the r e a c t i v i t y of model molecules such as acetylene could give insights into the manner i n which such unsaturated molecules bind to t r a n s i t i o n metal s u l f u r systems. Much data point to an intimate connection between H2 and the N2 binding s i t e i n nitrogenase. Simpson and Burris(58) confirmed the important finding of Hadfield and Buien(59) by showing that one H2 i s evolved for each N2 "fixed" even at 50 ATM N 2 , which i s well above the pressure of N2 at which saturation occurs. H2 evolution i s therefore a mandatory part of the N2 f i x a t i o n process(58-62) whose stoichiometry must be written as:


2

N2 + 8 H+ + 8 e-

> 2 NH3 + H2

Additionally, Wang and Watt have shown that the FeMo protein alone can act as an uptake hydrogenase(63). S p e c i f i c a l l y , H2 i n the presence of [FeMo] causes the reduction of o x i d i z i n g dyes such as methylene blue or dichlorophenolindophenol i n the absence of Fe protein. The hydrogen evolution and uptake behavior of nitrogenase proteins forces us to consider the ways i n which hydrogen can interact with t r a n s i t i o n metal s u l f u r centers. This we discuss i n the following section. Insights from Inorganic R e a c t i v i t y Many recent studies on inorganic systems that do not d i r e c t l y model the nitrogen f i x a t i o n process, nevertheless give p o t e n t i a l insight into b i o l o g i c a l nitrogen f i x a t i o n . We discuss two categories of relevant chemistry; f i r s t , acetylene binding and r e a c t i v i t y and second, dihydrogen binding and a c t i v a t i o n . C l a s s i c a l l y , acetylene binds to metal centers by using i t s ?r and w* o r b i t a l s to form, respectively, a-donor and jr-acceptor bonds to the metal. This s i t u a t i o n can hold when the metal i s i n a sulfur coordination64-65) environment such as i n MoO(S2CNR2)2(R(HCR)(64), and Mo(S2CNR2)2(RC=CR)2(65) Here, acetylene interacts d i r e c t l y with the metal center; a mode of binding that must be considered for nitrogenase substrates. The work of M. Rakowski DuBois and coworkers(66,67) points to a t o t a l l y d i f f e r e n t mode of acetylene binding. For example, (Cp')2Mo2S4 reacts with acetylene to produce

(Cp')2Mo2


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383


containing a bridging ethylene-1,2-dithiolate. In t h i s case, the acetylene has bound d i r e c t l y to the s u l f u r atoms i n the molybdenum s u l f u r complex. In related studies, acetylenes or substituted (activated) acetylenes are found to displace ethylene from bridging or terminal 1,2-dithiolate ligands(67.68). These reactions present examples of where the s u l f u r s i t e s rather than the metal s i t e s are reactive towards small unsaturated molecules of the c l u s t e r . A f i n a l example i l l u s t r a t e s the v e r s a t i l i t y that t r a n s i t i o n metal sulfur systems may provide. An activated acetylene has been shown(68) to insert into a metal-sulfur bond i n Mo202S2(S2)2^~ forming a v i n y l d i s u l f i d e chelating ligand,

on an M02O2S2 core(68). The r i c h chemistry of acetylene reacting with t r a n s i t i o n metal sulfur systems i s summarized i n Figure 3 which c l e a r l y suggests a number of p o s s i b i l i t i e s for nitrogenase reactivity. S i m i l a r l y , recent years have brought new insights into the way dihydrogen can be bound at a t r a n s i t i o n metal s i t e . Kubas and others(69,70,71) have shown that H2 i t s e l f can form simple complexes with a v a r i e t y of t r a n s i t i o n metal s i t e s i n which the H-H bond i s largely maintained. This finding contrasts with the c l a s s i c a l s i t u a t i o n i n which H2 interacts with a t r a n s i t i o n metal s i t e by oxidative addition to form a dihydride complex(72). In c e r t a i n cases the dihydrogen and dihydride complexes e x i s t i n simple equilibrium:(71) H M-«—N = N + H2

M -4

H ^

w

M

H

H

Crabtree(73) has suggested that the presence of a dihydrogen complex i s required for H2 to be displaced by N2 to form a dinitrogen complex. This reaction would explain the required stoichiometry of N2 reduction and H2 evolution. In other studies, Rakowski DuBois et al.(67) have shown r e a c t i v i t y of (Cp')2Mo2S4 with H2 to form (Cp')2Mo2S2(SH)2 i n which the dihydrogen i s cleaved but shows no d i r e c t i n t e r a c t i o n with the metal center. The r e s u l t i n g complex contains bridging SH groups but no d i r e c t metal-H bonding.(67) F i n a l l y , i n recent work, Bianchini et al.(73) reported that the dinuclear rhodium-sulfur complex {RhS[P(C6H5)2CH2CH2]3CH)2 reacts with two equivalents of H2 to y i e l d a complex (Rh(H)(SH)[P(C6H5)2CH2CH2]3CH}2 i n which two SH groups bridge the two Rh centers each of which contains a single hydrido ligand. Figure 4 i l l u s t r a t e s some of these p o s s i b i l i t i e s for hydrogen a c t i v a t i o n . Clearly, as i n the case for acetylene r e a c t i v i t y , metal-based, S-ligand based and M-S based r e a c t i v i t y


384



H H

H \

/

S ,

i

C=C

/

CONVENTIONAL TT-BONDING

DITHIOLENE COORDINATION

VINYL DISULFIDE

Figure 3. Possible modes of acetylene binding to t r a n s i t i o n metal sulfur s i t e s .

M ( X

i

DIHYDROGEN

M

x

\

„

DIHYDRIDE

ri' \

N

I

H

THIOL-HYDRIDE

%

M \

M

/

DITHIOL

Figure 4. Possible modes of hydrogen activation on t r a n s i t i o n metal sulfur s i t e s .


18, STIEFEL ET AL.


385


must each be considered as p o s s i b i l i t i e s for the hydrogen (or substrate) a c t i v a t i o n process of nitrogenase. The type of a c t i v a t i o n process that i s at work i n the enzyme i s currently unknown. Clearly we need greater s t r u c t u r a l d e f i n i t i o n of the active s i t e and t h i s w i l l be forthcoming through the continued application of sophisticated d i f f r a c t i o n and spectroscopic probes. D i f f r a c t i o n alone, however, w i l l be incapable of locating protons and possibly other low M.W. ligands. Therefore, spectroscopic probes such as ENDOR(32), and ESEEM(75-78), which are based on EPR spectroscopy, may become c r u c i a l i n elucidating mechanistically s i g n i f i c a n t s t r u c t u r a l d e t a i l s . In the remaining section, we b r i e f l y discuss the r e s u l t s of our recent studies using electron spin echo spectroscopy. Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy ESEEM i s a pulsed EPR technique which i s complementary to both conventional EPR and ENDOR spectroscopy(74,75). In the ESEEM experiment, one selects a f i e l d (effective g value) i n the EPR spectrum and through a sequence of microwave pulses generates a spin echo whose i n t e n s i t y i s monitored as a function of the delay time between the pulses. This r e s u l t i n g echo envelope decay pattern i s amplitude modulated due to the magnetic i n t e r a c t i o n of nuclear spins that are coupled to the electron spin. Cosine Fourier transformation of this envelope y i e l d s an ENDOR-like spectrum from which nuclear hyperfine and quadrupole s p l i t t i n g s can be determined. In nitrogenase, the S - 3/2 signal associated with FeMo-co provides the opportunity for d e t a i l e d ESEEM investigation76.77). Such investigations w i l l be presented i n d e t a i l elsewhere(78). but here we summarize some of the q u a l i t a t i v e r e s u l t s . In p a r t i c u l a r , we f i n d that N hyperfine s p l i t t i n g i s observed i n the ESEEM of the M center (FeMo-co i n [FeMo]). This l^N s p l i t t i n g i s not from the substrate, or from an intermediate or product of nitrogen f i x a t i o n since allowing the enzyme to turn over under 15^2 does not remove the s p l i t t i n g . Nor does the elimination of N-containing buffers remove the s p l i t t i n g . We conclude that the observed l ^ N - s p l i t t i n g i s due to a nitrogen atom associated with FeMo-co i n the protein. This nitrogen i s either part of the cofactor i t s e l f or i s provided to the cofactor from a protein side chain. To d i s t i n g u i s h these p o s s i b i l i t i e s ESEEM was c a r r i e d out on FeMo-co removed from the protein i n NMF solution. ESEEM of FeMo-co does not show the nitrogen quadrupole frequencies observed for the M center i n the protein. This shows that the observed s p l i t t i n g i s l i k e l y due to an l^N atom on the protein. Further, this nitrogen i s probably not of the deprotonated amide type, which i s the mode i n which NMF i s thought to bind to FeMo-co. Interestingly, the s p l i t t i n g parameters for the nitrogen are quite s i m i l a r to those involving imidazole nitrogen d i r e c t l y bound to low-spin heme(79). We conclude that a protein N binds d i r e c t l y to FeMo-co i n the FeMo protein. This conclusion i s reinforced by the determination of a non-zero i s o t r o p i c hyperfine term for the s p l i t t i n g , which unequivocally shows d i r e c t (Fermi 1 4


386


contact) interaction of the l^N nuclear and electron spins. Time should reveal the p a r t i c u l a r significance that this nitrogen has for the structure or functioning of the M center.


Conclusion Although we know a great deal about nitrogenase, we s t i l l have much to learn i n this pre-structural phase of nitrogenase enzymology. We do not yet know the structure of any of the metal clusters i n the FeMo or FeV proteins. Nor do we know the arrangement of these clusters i n the proteins. However, even with s t r u c t u r a l d e f i n i t i o n , which i s i n progress(80-82), we w i l l have to continue to apply the most powerful tools of physical bioinorganic chemistry to determine how hydrogen and substrates are handled by this remarkable enzyme. Acknowledg ments We thank Drs. Graham George and Roger Prince and the reviewers f o r useful comments and are grateful to Drs. Brian Hales and Graham George f o r informing us of results p r i o r to publications. Literature Cited 1. 2.

Blackburn, T. H. i n Microbial Geochemistry; W. E. Krumbein, Editor, Blackwell S c i e n t i f i c , Oxford, 1983; p 63. Postgate, J . R. Fundamentals

of Nitrogen

Fixation;

Cambridge

University Press, Cambridge, New York, 1982. 3.

Hardy, R. W. F. Treatise

on Dinitrogen

Fixation;

Wiley and

Sons, New York, 1979; Section I. 4.

B r i l l , W. J . NATO Adv.

5. 5a.

Haselkorn, R. Ann. Rev. Microbiol. 1986, 40, 525. Robinson, A. C.; Dean, D. R.; Burgess, B. K. J. Biol. Chem. 1987, 262, 14,327. Stephens, P. J . i n Molybdenum Enzymes; T. G. Spiro, Editor; J . Wiley and Sons, 1985; p 117. Gibson, A. H.; Newton, W. E., Editors, Current Perspectives in Nitrogen Fixation; Australian Academy of Science, Canberra, 1981. Veeger, C.; Newton, W. E., Editors, Advances in Nitrogen Fixation Research; Nijhoff/Junk, The Hague, 1984. Evans, H. J . ; Bottomley, P. J . ; Newton, W. E., Editors,

6. 7.

8. 9.

Nitrogen

Fixation

Sci.

Research

Inst.,

Progress;

10.

1985. S t i e f e l , E. I. i n ref. 7, p 55.

11.

Hageman, R. V; Burris, R. H. Proc.

Ser.

A,

1983, 63,

231.

Martinus N i j h o f f ,

Natl.

Acad.

Sci.

Boston,

USA 1978,

75, 2699. 12. Sundaresan, V.; Ausubel, F. M. J. Biol. Chem. 1981, 256, 2808. 12a. Hausinger, R. P.; Howard, J . B. J. Biol Chem. 1983, 258, 13,486. 13. Orme-Johnson, W. H.; Davis, L. C.; Henzl, M. T.; A v e r i l l , B. A.; Orme-Johnson, N. R.; Münck, E.; Zimmerman, R. Recent Developments

in Nitrogen

Fixation;

W. E. Newton, J . R.


18. STIEFEL ET AL.

14. 15. 16.

18.


19. 20.

22.

24. 25. 26. 27.

29. 30.

31. 32.

250.

J.

Biochem.

1985,

148,

Druzhinin, S. Y.;

34. 35. 36. 37.

Uznskaya, A. M. Dokl.

Akad.

Nauk SSR

1978,

537,

185.

McLean, P. A.; Papaefthymiou, V.; O'Hagen, F.; Orme-Johnson; Münck, E. J. Biol. Chem. 1987 262, 12,900. Kurtz, D. M.; McMillan, R. S.; Burgess, B. K.; Mortenson, L. E.; Holm, R. H.; Proc. Nat. Acad. Sci. USA, 1979, 76, 4986. Venters, R. A.; Nelson, M.; McLean, P. A.; True, A. E.; Levy, M. A.; Hoffman, B. M.; Orme-Johnson, W. H.; J. Am. Chem. Soc. 1986, 108, 3487. Shah, V.; Brill, W. J . Proc. Nat. Acad. Sci. USA 1977, 74, 3249. Lough, S. M.; Jacobs, D. L.; Lyons, D. M.; Watt, G. D.; McDonald, J . W. Biochem.

33.

499.

1981, 262, 1177. Thorneley, R. N. F.; Lowe, D. J . i n Molybdenum Enzymes T. G. Spiro, Editor; J . Wiley and Sons, New York, 1985; p 221. Brigle, K. E.; Newton, W. E.; Dean, D. R. Gene 1985, 37, 37. Dixon, R. A. J. Gen. Micro. 1984, 130, 2745. Watt, G. D.; Burns, A.; Tennent, D. L. Biochemistry 1981, 20, 7272; Watt, G. D.; Wang, Z. C.; Biochemistry 1986 25, 5196. Zimmermann, R.; Münck, E.; Brill, W. J . ; Shah, V. K.; Henzl, M. T.; Rawlings, J . ; Orme-Johnson, W. H. Biochim. Biophys. Acta.

28.

189,

Mortenson, L. E.; Thorneley, R. N. F. Am. Rev. Biochem. 1979, 48, 387. Kulikov, A. V.; Syrtsova, L. A.; Likhtenshtein, G. I.; Popko, E. V.;

23.

1986,

Morgan, T. V.; Prince, R. C.; Mortenson, L. E. FEBS Letters 1986, 206, 4. Carney, M. J . ; Holm, R. H.; Papaefthymiou, G. C.; Frankel, R. B. J . Am. Chem. Soc. 1986, 108, 3519. Noodleman, L.; Norman, J . G., J r . ; Osborne, J . H.; Aizman, A.; Case, D. A. J. Am. Chem. Soc. 1985, 107, 3418. Watt, G. D.; Wang, Z.-C; Knotts, R. R. Biochemistry 1986, 25, 8156; Cordewener, J . ; Haaker, H.; Van Ewijk, P.; Veeger, C. Eur.

21.

387

Postgate and C. Rodriguez Barrueco, Editors; Academic Press, 1977, p 131. Lindahl, P. A.; Day, E. P.; Kent, T. A.; Orme-Johnson, W. H.; Münck, E. J. Biol. Chem. 1985, 260, 11160. Watt, G. D.; McDonald, J . W. Biochemistry 1985, 24, 7226. Hagen, W. R.; Eady, R. R.; Dunham, W. R.; Haaker, H. FEBS Letters

17.


Biophys.

Res.

Comm. 1986,

139,

740.

S t i e f e l , E. I.; S. P. Cramer i n Molybdenum Enzymes; T. G. Spiro, Editor; J . Wiley and Sons, 1985, p 88. Walters, M. A.; Chapman, S. K.; Orme-Johnson, W. H. Polyhedron 1986, 5, 561. McLean, P. A.; Dixon, R. A.; Nature, 1981, 292, 655. McLean, P. A.; Smith, B. E.; Biochem. J. 1983, 211, 589. Hawkes, T. R.; McLean, P. A.; Smith, B. E. Biochem. J. 1984, 217, 317.

38.

Bortels, H. Arch.

39.

Burgmayer, S. J . N.; S t i e f e l , E. I. J. 943.

Mikrobiol.,

40.

Bortels, H. Zentbl.

Bakt.

1930,

Parasiten

1,

Abt.

333.

Chem. Educ.; II

1935,

1985, 62, 95,


193.

388 41.

METAL CLUSTERS IN PROTEINS McKenna, C. E.; Benemann, J . R.; Traylor, T. G. Biophys.

42.

Res.

Comm. 1971, 42, 353.

Benemann, J . R., McKenna, C. E.; L i e , R. F.; Traylor, T. G.; Kamen, M. D. Biochim.

44. 45.


46. 47. 48.

50. 51.

53.

54. 55.

57.

1987, 26, 1795.

1987, 327,

167.

Dilworth, M. J . Recueil

Travaus

Chim.

des

Pays-Bas

1985, 24, 273.

Simpson, F. B.; Burris, R. Science 1984, 224, 1095. Hadfield, K. L.; Bulen, W. A. Biochemistry 1969, 8, 5103. S t i e f e l , E. I.; Newton, W. E.; Watt, G. D.; Hadfield, K. L.; Bulen, W. A. Advances Bioinorganic Chemistry

61.

des

1987, 106, 175. Burgess, B. K. i n Molybdenum Enzymes; T. G. Spiro, Editor; J . Wiley and Sons, New York, 1985; p 161. Rubinson, J . F.; Burgess, B. K.; Corbin, J . L.; Dilworth, M. J . Biochemistry

58. 59. 60.

1972, 264, 25.

Morningstar, J . E.; Hales, B. J . J. Am. Chem. Soc. 1987, 109, 6854. Arber, J . M.; Dobson, B. R.; Eady, R. R.; Stevens, P.; Hasnain, S. S.; Garner, C. D.; Smith, B. E. Nature 1987, 372, 325. George, G. N.; Cramer, S. P.; Hales, B. J . ; personal communications, submitted for publication. Eady, R. R.; Robson, R. L.; Smith, B. E.; Lowe, D. J . ; M i l l e r , R. W.;

56.

Acta,

Robson, R. L.; Eady, R. R.; Richardson, T. H.; M i l l e r , R. W.; Hawkins, M. H.; J . R. Postgate Nature 1986, 322, 388. Eady, R. R.; Robson, R. L.; Richardson, T. H.; M i l l e r , R. W.; Hawkins, M. Biochem. J. 1987, 244, 197. Dilworth, M. J . ; Eady, R. R.; Robson, R. L.; M i l l e r , R. W. Nature

52.

Biophys.

Bishop, P. E.; J a r l e n s k i , D. M. L.; Hetherington, D. R. Proc. Nat. Acad. Sci. USA, 1980, 77, 7342. Bishop, P. E., Premakumar, R.; Dean, D. R.; Jacobson, M. R.; C h i s n e l l , J . R.; Rizzo, T. M.; Kopczynski, J . Science 1986, 232, 92. Hales, B. J . ; Langosch, D. J . ; Case, E. E. J. Biol. Chem. 1986, 261, 15301. Hales, B. J . ; Case, E. E.; Morningstar, J . E.; Dzeda, M. F.; Mauterer, L. A. Biochemistry 1987, 26, 1795. Morningstar, J . ; Johnson, M. K.; Case, E. E.; Hales, B. J . Biochemistry

49.

Biochem.

Comm. 1970, 41, 1501.

Burns, R. C.; Fuchsman, W. H.; Hardy, R. W. F. Biochem. Biophys.

43.

Res.

in Chemistry Series, II; 1977, p. 353.

No.

162,

Burgess, B. K.; Wherland, S.; Newton, W. E.; S t i e f e l , E. I. Biochemistry

1981, 20,

5140.

62.

wherland, S.; Burgess, B. K.; S t i e f e l , E. I.; Newton, W. E.

63.

Wang, Z.-C.; Watt, G. D. Proc. Natl. Acad. Sci. USA, 1984, 81, 376. Newton, W. E.; McDonald, J . W.; Corbin, J . L.; Ricard, L.;

Biochemistry

64.

1981, 20,

Weiss, R. Inorg.

65. 66.

5132.

Chem. 1980, 19,

1997.

Herrick, R. S.; Templeton, J . L. Organometallics, 1982, 1, 842. Rakowski DuBois, M.; R. C.; Haltiwanger, R. C.; M i l l e r , D. J . ; Glazmaier, G. J. Am. Chem. Soc; 1979, 101, 5245.


18. STIEFEL ET AL. 67.

68. 69. 70. 71. 72.


389

Rakowski DuBois, M.; VanDerveer, M. C.; DuBois, D. L.; Haltiwanger, R. C.; M i l l e r , W. K. J. Am. Chem. Soc. 1980, 102, 7456. Halbert, T. R.; Pan, W.-H.; S t i e f e l , E. I. J. Amer. Chem. Soc. 1983, 105, 5476. Kubas, G. J . ; Ryan, R. R.; Swanson, B. I.; Vergamini, P. J . ; Wasserman, H. J . J. Am. Chem. Soc. 1984, 106, 451. Kubas, G. J . ; Ryan, R. R. Polyhedron 1986, 5, 473. Kubas, G. J . ; Unkefer, C. J . ; Swanson, B. I.; Fukushima, E. J . Am. Chem. Soc. 1986, 108, 7000. Cotton, F. A.; Wilkinson, G. Advanced

Inorganic

Chemistry;

J.


Wiley and Sons, 1980. 73.

Crabtree, R. H. Inorg.

74.

Bianchini, C.; M e a l l i , C.; Meli, A.; Sabat, M. Inorg. 1986, 25, 4617.

Chim.

Acta

75.

Mims, W. B.; Peisach, J.

Biological

1986, 125,

Magnetic

L7.

Chem.

Resonance;

Vol.

3; J . Berliner and J . Reuben, Eds.; Plenum, New York, 1981; p 213. 76.

77.

78. 79. 80.

Mims, W. B.; Peisach, J . Biological

G. J.

81. 82.

Applications

of

Magnetic

Resonance; R. G. Shulman, Editor; Academic Press, 1980; p 221. Orme-Johnson, W. H.; Lindahl, P.; Meade, J . ; Warren, W.; Nelson, M.; Groh, S.; Orme-Johnson, N. R.; Münck; Huynh, B. H.; Emptage, M.; Rawlings, J . ; Smith, J . ; Roberts, J . ; Hoffman, B.; Mims, W. B. i n ref. 8, p 79. Thomann, H.; Morgan, T. V.; Burgmayer, S. J . N.; Bare, R. E.; J i n , H.; S t i e f e l , E. I. J. Am. Chem. Soc., 1987, 109, 7913. Peisach, J . ; Mims, W. B.; Davis, J . L. J. Biol. Chem. 1979, 254, 12,379. Yamane, T.; Weininger, M. S.; Mortenson, L. S.; Rossmann, M. Biol.

Chem. 1982, 257,

1221.

Rees, D. C.; Howard, J . B. J. Biol. Chem. 1983, 258, 12,733. Sosfenov, N.; Adrianov, V. I.; Vagin, A. A.; Strokopytov, B. V.; Vainstein, B. K.; Shilov, A. E.; Gvozdev, R. I.; Likhtenshtein, G. I.; Mitsova, I. Z.; Blazhchuk, I. S. Dokl. Akad. Nauk. SSR 1986, 291, 1123.

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