A survey of transition metal dinitrogen complexes

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Authors Onsgard, Henry Adolph, 1945-

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A SURVEY OF TRANSITION METAL

DINITROGEN COMPLEXES

by

Henry A. Onsgard

A Thesis Submitted to the Faculty of the

DEPARTMENT OF CHEMISTRY..

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

1 9 7 1

STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of re­quirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library„

Brief quotations from this thesis are allowable without special permissiony provided that accurate acknowledgment of source is made* Requests for permission for extended quotation from or reproduction of this manuscript in.whole or in part may be granted by the head of the major department or the Dean of The Graduate College when in his judg­ment the proposed use of the material is in the interests of scholar­ship. In all other instances, however, permission must be obtained from the author.

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

R A J ' i ) 7 / M a m .....ROBERT D. FELTHAM

Associate Professor of Chemistry

ACKNOWLEDGMENT

I wish to thank Dr. Robert D. Feltham for his help and guid­

ance* Special thanks go to Dr. Philip G. Douglas for helpful dis­

cussions.

I am grateful to the Department of Chemistry, The University

of Arizona, for the teaching assistantships.

' . - \ : . . .

TABLE OF CONTENTS

Page

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I - INORGANIC DINITROGEN COMPLEXES......... ... . 1

INTRODUCTION . 2

PREPARATIVE METHODS . . . . . . . . . . . . . . . . . . , . . . . 3

Method I: Hydrazine e . » „ » . „ , » . » « ............ 3Method II: Azides „ , . e 5Method III: Gaseous Nitrogen . . 6Method IV: Reduction of Metal Acetylacetonates With

Metal Alkyls c , » ..............« « • • • • • • • • • ' 8Method V: Metal Reduction in the Presence of Nitrogen . » . 10

. Method VI: Nitrous Oxide Reduced to Dinitrogen . . . . . . . 12Method VII: Nitrous Acid on Ammonia 12Method VIII: Dehydrogenation of Ammonia ..................... 13Method IX: N0+ With A z i d e . * 13Summary „ . . . , , » . . . . » . .. . « » ............ .. 13

INORGANIC MODELS FOR NITROGENASE . . . . . . . . . . . . . . . . 14

J. Chatt Model ....................... 14G, Schrauzer Model ........... . 15

PART II - ENZYMATIC NITROGEN FIXATION SYSTEMS . . . . . 16

INTRODUCTION ........................... 17

A symbiotic.................................. 17"Azotobacter Vinelandii" . . . . . . . 17"Clostridium Pasteuriamum"....................... 18

Symbiotic K .................... 19

ROLE OF METAL IONS IN ENZYMATIC NITROGEN FIXATION.............. . 20

Presence of Metals ................... 20Mechanism and Intermediates . . . . . . . . . . . . 22

TABLE OF CONTENTS— Continued

Page

POSSIBLE ENZYME MODELS ............ 25

PART III - CONCLUSION ................ 28

RESUME . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 30

ABSTRACT

Since the discovery of the first dinitrogen complex in 1965

by A, D. Allen and C. V. Senoff, many different dinitrogen complexes

have been formed by nine different preparative methods. This review

deals with the different preparative methods of dinitrogen complexes,

inorganic models for nitrogenase, enzymatic nitrogen fixation systems,

and possible enzymatic model. The review covers the literature to

July 22, 1970.

PART I .

INORGANIC DINITROGEN COMPLEXES

1

INTRODUCTION

A. D. Allen and C. V. Senoff discovered, in 1965, the first

dinitrogen (Chatt, Dilworth, Gunz et al. 1970) complex. Since this

discovery, dinitrogen transition metal complexes have been of great in­

terest to the inorganic chemists. Since the thirties, biochemists have

been examining the problem of enzymatic nitrogen fixation. However,

only recently have they been able to isolate metalloenzyme systems such

as nitrogenase,which would convert gaseous nitrogen to ammonia under

mild conditions (Murray and Smith 1968, Hardy and Burns 1968). Current­

ly, several inorganic chemists are preparing dinitrogen complexes, which

may serve as models for nitrogenase.

The number of dinitrogen complexes and their methods of prepara­

tion are rapidly increasing. Nine routes are now known for the prepa­

ration of isolatable dinitrogen complexes. Stable dinitrogen complexes

have been formed with the following transition metals: tungsten,

ruthenium, molybdenum, osmium, n i c k e l , iron, iridium, rhodium, rhenium,

and cobalt.

Stable dinitrogen complexes are characterized by having the

central metal atom in a low oxidation state (itzkovitch and Page 1968)

(i.e., high electron density) and with strong electron donating ligands

(to preserve the high electron density). For example, the Ru(ll) d ini­

trogen complex, [ R u ^ H ^ t d ^ ] ^ * , when oxidized to Ru(lll), thereby re­

ducing the electron density on the ruthenium, loses nitrogen.

2

PREPARATIVE METHODS

Described below are five general methods of producing dinitro­

gen complexes.

Method I: Hydrazine

The first method of preparing a dinitrogen complex was de­

scribed by Allen and Senoff (1965). The method consists of reacting

ruthenium trichloride with hydrazine h y d r a t e :

RuCl + N 2H 4 -H2 0 ------ - [Ru (NH3 )5N 2 ]2+ (1)

I

Dinitrogen complexes of this type have been isolated with Cl , Br , I ,

BF^- , and PF^ , as the counter anion. The nitrogen-stretching frequency

observed by Allen and Senoff (1965) was in the range of 2070-2115 cm \

depending upon the anion. However, there was a problem of purification

with this method. A hydrazine impurity caused some false properties to

be ascribed to the dinitrogen complex (Chatt and Richards 1968). The

reactions of (NH4 ) (RuCl ), (CH S03 ) [ ( N H ^ R u O ^ O ) ] , and K ^ R u C l ^ O )

with hydrazine hydrate also produce compound I (Allen et al. 1967).

This method has been used for the preparing of rhenium and osmium di­

nitrogen complexes and the osmium and ruthenium bis(dinitrogen) (Chatt,

Dilworth, Gunz et al. 1970) complexes. Rhenium(l) dinitrogen complexes

(Moelwyn-Hughes and Garner 1969) have been prepared by the following

m e t h o d :

4N 9 H 4Re(C0)4 LX — Re(CO)3 (NH )(N )L (2)

L = P H e ^ P h , AsMe^Ph X = Br

andn 9h

Re(CO)3X L 2 — Re(CO)2 (NH2 )(N2 )L2 (3)

L = PMe Ph X = Cl

The osmium complexes were prepared by Allen and Stevens (1967),

(NH4)20 sC16 + N2H4 *H20 -r— --u-£- [Os (NH3)5N2]X2 (4)

and by Chatt (1969),

OsCl3(PBu Ph)3 + N2H4 ----- ^ O sC12N2(PBu 2PH)3 (5)

Products of (4) and (5) could not be purified. Both the dinitrogen and

bis(dinitrogen) complexes of osmium were obtained from the following r e ­

actions (Allen and Stevens 1967, Borod'ko and Shilov 1969):

K2°sC16or

OsCl4 + N2H4 — *-[OsN2(NH3)5]X2 + [Os(N2)2(NH3)4]X2 (6)or

OsCl OH 1

By carrying out the reaction of Allen and Senoff (1965) at low tempera­

ture (-23°), [Ru(NH3 )4 (N2 )2 ]Br2 was isolated from the reaction mixture

(Fergusson and Love 1969).

Method II; Azides

There are several preparations of dinitrogen complexes reported

involving azides (azide decomposition, azide plus acid, and acyl azide).

Only ruthenium dinitrogen complexes have been prepared from the decomp­

osition of azides. Allen et a l . (1967) f o u n d :

H,0Ru(NH ) (H 0)(CH SO ) + NaN^ — ^ - ^ [ R u C N H j ) ^ ] (7)

producing compound I. A bis-azido complex of ruthenium,after thermal

decomposition, gave an azido-dinitrogen. When this compound was

treated with nitrous acid, a bis(dinitrogen) complex was formed. The

reactions (Kane-Maguire et al. 1968) can be summarized:

° HNO[Ru(en)2 (N3 )2 ]PF6 [Ru(en)2 (N2 )N3 ]PF^ ------------------(8)

[Ru(en)2 (N2 )2 ]2+

A dinitrogen-bridged ruthenium complex (Kane-Maguire, Basolo, and P ear­

son 1969) has been formed by

H O ,[Ru (NH3 )5N 3 1(N3 )2 rRu(NH3 )5N 2 ]Z+ + (9)

II

The reaction is postulated to involve a protonated nitrene intermed­

iate. The acyl azide preparation has been used to prepare dinitrogen

complexes of iridium and rhodium. Collman (jj}_ Collman and Kang 1966,

Collman et al. 1968) found that:

RON L NN U L\ / C1IrC1(L)2C° CHCl- - ^ zIrx L a 0 )

L = PlyP R = Ph, NH C^H^O

(Ph^P) 2 Rh(N^)Cl was produced by means of the same method (Ukhin,

Shvetsov, and Khidekel 1967). In a closely related method, Cha t t , M e l ­

ville, and Richards (1969) have produced a series of very stable rhenium

dinitrogen complexes. The reactions (Chatt, Dilworth, and Leigh 1969)

a r e :

L C|1 ,NN. , L C,1 .NNCOPhRe / Ph — -— ^ Re ^ Re C l ( N 0 )L0L 0 (11)

L - l - O 7 L ' cl-L-

L = PPH^ L ' = phosphine

and (Chatt, Dilworth, and Leigh 1970)

■ V > ' > \ Ph pv £ " !Ph 3P CI1X 0 / Ph3p/C X c 0

0

Method III: Gaseous Nitrogen

A significant number of dinitrogen complexes have been prepared

from gaseous nitrogen by ligand displacement or by the dinitrogen fill­

ing a vacant coordination site.

The complex, FeH^L^ (L = PEtPh^ or PBuPh^), readily reacts with

nitrogen at room temperature and atmospheric pressure whether in solu­

tion or in the solid state. The reaction (Sacco and Aresta 1968) is:

I

7NaBH, N

FeCl + 3L -------- ^ FgH2L3 FeH2N2L3 (13)

Iron dinitrogen complexes have also been prepared by chloride displace­

ment from the coordination sphere by nitrogen (Bancroft, Mays, and

Prater 1969).

NaBPh,FeHClL2 + N ------- =*- FeHN^L BPh^ (14)

From the following reaction, hydrogen can be displaced by n i ­

trogen from CoH^L^ to give a dinitrogen-cobalt complex (Sacco and Rossi

1967; Rossi and Sacco 1969; Lorbeth, Noth, and Rinze 1969):

CoH Lg + N2 CoH(N2 )L3 +-H2 (15)

(L = PPh_, PEtPh2 , PB u 3 , PMePh2 )

Another hydrogen displacement reaction (Bell, Chatt, and Leigh 1970a)

i s :

O sH4 L 3 + N 2 ----------- O sH 2N 2L 3 (16)

An example of nitrile displacement by nitrogen (Misono et al. 1969) is:

CoH(RCN)(PPh3 )3 + N CoHN2 (PPh3 )3 + RCN (17)

R = Me or Et

Under mild conditions, it is possible to form compound I and II

by replacing water from the coordination sphere of [Ru (NH3 )3H 20]^+ with

gaseous nitrogen. The reactions (Allen and Bottomley 1968a; Harrison

and Taube 1967; Harrison, Weissberger, and Taube 1968) a r e :

N[Ru (NH3 )5H 20]2+ [Ru (NH3 )5N 2 ]2+ + [Ru 2 (NH3 )1()N 2 ]4+ (18)

Reaction (18) has been studied calorimetrically. A H for the overall

reaction is -22 — 2 kcal/mole (Farquhart, Rusnock, and Gill 1970),

Gaseous nitrogen is capable of displacing a phosphine ligand in

the reaction (ito et al. 1970):

N HH Rub H 0RuL_N. m H.RuL. (19)I 4 1 5 1 N2 4 5

Method IV; Reduction of Metal Acetylacetonates With Metal Alkyls

Transition metal acetylacetonate (acac) complexes react with

aluminum organometallics and gaseous nitrogen in the presence of a

phosphine ligand. By this method, a molybdenum bis(dinitrogen) com­

plex (Hidai et al. 1969) was f o r m e d :

Mo(a c a c ) 3 + ( i s o B u ^ A l + (Ph2P C H 2 )2 + N 2

z \ /PXC|H2 X y C|H2CH, ,M°\ CH (20)

N / N 2

Dinitrogen complexes of Co and Ni, including a bridged dinitrogen Ni

complex, have been prepared using this method (Yamamoto et al. 1967):

Co(acac)3 + 2AlEt2OEt + 3PPh3 + Ng -----^ (Ph3P ) 3CoN2 (21)

I b e r s 1 group (jji Enemark et al. 1968) has carried out an X-ray struc­

tural analysis of the dinitrogen complex obtained from the reac t i o n :

Co(acac)^ + AlEt^ + PPh^ +Et20

CoH(N )(PPh3 )3 *Et20(nBu)20

CoH(N )(PPh3 )3

III

(22)

Tlie hydride is located in the axial position trans to the dinitrogen

(Davis, P a y n e , and Ibers 1969). To prove that compound III was a hy-

pound III. Nineteen hydrogens (all of the ortho-hydrogens of the

aromatic rings and the hydride) per mole of the cobalt dinitrogen com­

plex exchanged with gaseous deuterium. To understand the exchange r e ­

actions of cobalt dinitrogen complexes the following reaction scheme is

useful (Allen and Bottomley 1968a).

dride, Parshall (1968) carried out a deuterium exchange with D2 on com-

-H 2 -N 2

(Ph3P)3CoH3 (Ph3P) H,3 ' 2 ^ / 2Co

Thus it is believed that compound III is a hydride. It is postulated

that only the hydride is formed in the following reaction (Misono,

U c h i d a , and Saito 1967; Misono, Uchida, Saito, and Song 1967):

10

CoCacac)^ + 3PPh^ + + Al(isoBu)^ N^CoCPPh^)^

+ N CoH(PPh3 )3 (23)

The nickel dinitrogen complex is formed by (Srivastava and Bigorgne

1969):o

NiCacac)^ + ^ 2 ^ 5 ^ 3 ^ + (isoBu)3Al ------ *-

H N i ( N 2 )[(C2H 5 )3P]2 (24).

The bridging dinitrogen complex is formed by (Jolly and Jonas 1968):

Ni(acac)2 + 4 (C6H 1 1 )3P + 4 A l(CH3 )3 + Ng

N 2 (Ni[p(C6H 1 1 )3 ]2 )2 (25)

In a closely related reaction a triphenyIphosphine complex of

ruthenium is used as the starting m a t e r i a l . The reaction (Knoth 1968)

i s :

(Ph3P) 3RuHCl + Et3Al + N 2 ----------- (Ph3P ) 3Ru(N2 )(H)2 (26)

Method V: Metal Reduction inthe Presence of Nitrogen

Several complexes have been reduced by amalgamated zinc and re­

acted with gaseous nitrogen to produce a dinitrogen complex. In 1966,

Shilov, Shilova, and B o r o d ’ko reported that the reaction of ruthenium

trichloride with amalgamated zinc plus gaseous nitrogen gave a dinitro­

gen complex, as evidenced by infrared absorption ( ^ N 2 2140 cm ̂ and

*^N2 2070 cm ^ ). H o w e v e r , the complex was not isolated. Allen and

Bottomley (1968b) reported the isolation of compounds I and II from the

r e a c t i o n :

11

Ru (NH3)5C1 Cl2 + Zn/Hg + ( [(NH3)10Ru2]N2)4+

+ [Ru (NH3 )5N 2 ]2+ (27)

Other ruthenium dinitrogen complexes have been formed by a similar r e ­

action (Harris and Wright 1970):

N Br Br [Ru(trien)Br0 ]Br [(trien)Ru-N0-Ru(trien)]^ +

Z “ 2 4

H O >-[Ru 2 (H 2 0 )2 (N 2 )(trien)^] -----[Ru(H 2 0 )2 (trien)]

+ [RuCH^O)(N^)(trien)]^+ (28)

Since then, an osmium dinitrogen complex has been formed by a similar

reaction (Chatt, Leigh, and Richards 1969):

mer OsX (PR^lg + Zn/Hg + OsX2N 2 (PR3 )3 (29)

X = Cl, Br

A tungsten dinitrogen complex (Bell, Chatt, and Leigh 1970b)

has been prepared by a sodium amalgum reaction which is

trans WCl.L cis W ( N 0 )0 (PMe_Ph), +4 L 2 Z Z 4

trans W(N ) (PhgPCH CH PPh2 )2 (30)

L = tertiary phosphine

Apart from the preceding general reactions, there are four

other reactions which are more specific in n a t u r e . The following

methods, VI through IX, deal with these individual reactions.

Method VI: Nitrous OxideReduced to Dinitrofien

One of the reactions discovered by Allen and Bottomley (1968b)

(amalgamated zinc reduction of [Ru(NH^)^Cl] Cl^ in the presence of n i t r o ­

gen) was used by Diamantis and Sparrow (1969). Both gaseous nitrogen

and nitrous oxide were used. The nitrous oxide was found to react

faster to give the desired products, compounds I and II. Recently the

intermediate in this reaction, [Ru(NH^) has been isolated

(Diamantis and Sparrow 1970). When the reaction is carried out with 15 15N NO or NNO two different metal dinitrogen-stretching frequencies

are observed for compound I. Upon standing, isomerization occurs to

give a mixture of two isomers. The isomerization mechanism appears toN 2 +

be monomolecular via the intermediate [(NH^)^Ru jjj] (Armor and Taube

1970).

Method VII: Nitrous Acid on Ammonia

Coordinated ammonia can also be converted to dinitrogen when

reacted with nitrous acid. An example of this reaction (Scheidegger,

Armor, and Taube 1968) is:

C(NH3 )50 sN 2 ]C12 + N a N 0 2 + HC1 ----- ^ [ ( N H ^ O s ^ ) ^ 24" (31)

or HNO

The mechanism is unknown but it is presumed to be analogous to the

diazotization of an amine.

13

Method VIII: Dehydrofienation of Ammonia

One of the most interesting methods of producing a dinitrogen

complex was found by J . Chatt and J. E. Fergusson (1968) when they tried

to purify [Ru(NH^)^J Cl^. Compound I is obtained by the r e a c t i o n :

K *1 H ORuCl + N H 3 ■ - [RU (NH3 )6 ] Cl2 — ^ [Ru (NH3 )5N 2 ]C12 + (32)

The nitrogen, therefore, is being produced by dehydrogenation of the

ammonia.

Method IX: N 0 + With Azide

A novel method of preparing dinitrogen complexes has recently

been discovered by P. G. Douglas, R. D. Feltham, and H. G. Metzger

(1970). This involves the reaction of a coordinated azide with N 0 + .

The reaction is:

RuN Cl(Das)2 + NOPF^ > - [ R u C l N 2 (Das)2 ]PF^ + ^ 0 (33)

Das = o^-phenylenebis(dimethylarsine)

Summary

Despite the preparation of numerous dinitrogen complexes, there

is as yet no standard procedure for their preparation. Such a procedure

would be helpful in the isolation and hence the understanding of these

compounds.

INORGANIC MODELS FOR NITROGENASE

From the understanding developed from the chemistry of inorganic

dinitrogen complexes, several inorganic models for enzymatic fixation

of nitrogen have been prop o s e d . Nitrogenase, a metalloenzyme system,

appears to have both iron and molybdenum ions directly involved in the

fixation p r o c e s s . Iron probably forms a dinitrogen complex, but the

role played by the molybdenum in this process is still uncertain.

J. Chatt Model

J. Chatt (iin Chatt, Dilworth et a l . 1969; Chatt, Dilworth,

Leigh et al. 1970) has postulated an inorganic model for nitrogenase.

The model involves the formation of nitrogen-bridged metal complexes.

Several nitrogen-bridged dimers of rhenium(l) were prepared by C h a t t .

The nitrogen-stretching frequency of these complexes decreased from

that of nitrogen gas ( L ^ = 2331 cm *) to lower values indicating a de­

crease in the nitrogen bond strength. This decrease in the nitrogen

bond strength may increase the ease of nitrogen reduction.

The model complexes were formed by a reaction of a rhenium di­

nitrogen complex with a transition metal chloride:

ReCl(N0 )(PMe0Ph)/ + MCI Cl(PMe0Ph), Re-NN-MCl2 2 4 x 2 4 x

M = Ti, x = 3; C r , x = 3; Mo, x = 4

The reduction of the nitrogen-stretching frequency was from 2331 cm *-1in gaseous nitrogen to 1680 cm in the case of molybdenum rhenium

14

15

nitrogen-bridged complex. This reduction corresponds to a decrease in

the bond strength of approximately 100 kcal/mole. This decrease in the

nitrogen bond strength may facilitate the reduction of the nitrogen.

G. Schrauzer Model

Schrauzer and Schlesinger (1970) have discovered that acetylene

is reduced by a molybdenum-thiol catalyst system. Other transition

metal thiol catalyst systems were also investigated. They were found

however to have little or no reductive ability when compared to the

molybdenum-thiol system. Schrauzer has suggested that this system is a

good model for nitrogenase.

The molybdenum-thiol system uses molybdenum (VI or V ) compounds

(Na^MoO^, MoO^, HoCl^, or polyheteromolybdates) and a wide variety of

thiols. The following thiols demonstrated a high activity: dithio-

erythritol, 1-thioglycerol, 2-mercaptoethanol, and cysteine. When the

molybdenum compound and the thiol were reacted, the highest catalytic

reduction activity for acetylene was observed at the metal/ligand ratio

of 1:1. From this discovery, Schrauzer (in Schrauzer and Schlesinger

1970) concludes that the nitrogenase enzyme system reduces nitrogen at

a molybdenum site in which the molybdenum is bound to sulfur ligands.

The molybdenum-thiol systems described above will reduce nitrogen but

only at elevated pressures.

PART II

ENZYMATIC NITROGEN FIXATION SYSTEMS

\

16

INTRODUCTION

The enzymatic nitrogen fixation systems can be grouped into two

categories (Murray and Smith' 1968): (l) asymbiotic (free-living micro-'

organisms) and (2) symbiotic (bacteria incorporated in the root systems

of certain plants). The mechanism of these enzymatic nitrogen fixation

systems is under intensive investigation. To investigate this mechanism,

active cell-free extracts have been isolated from these systems and

their properties studied.

Asymbiotic

Asymbiotic systems.are free living microorganisms, such as blue-

green algae, some yeasts, and bacteria. Most of the research on asym­

biotic systems has been done on cell-free extracts of two bacteria,

"Azotobacter Vinelandii11 (AV) (aerobic bacteria) and "Clostridium Pas-

teuriamum" (CP) (anaerobic bacteria) (Andriyuk 1967; Fay and Cox 1967;

Mortenson 1964; Nicholas 1963; Patil, Pengra, and Yoch 1967).

"Azotobacter Vinelandii"

The active cell-free extracts isolated from AV are stable in

the air and are able to reduce nitrogen to ammonia (Nicholas, Silvester,

and Fowler 1961). The source of the reducing power in the bacteria is

unknown; therefore, in the studies of these extracts, dithionite has

been used as the reducing agent. The studies (Murray and Smith 1968,

Hardy and Knight 1966, Dilworth et al. 1965) have shown that when the

- ■\

18extracts are mixed with ATP and dithionitev hydrogen is evolved. Hy­

drogen evolution slows when nitrogen is also added to the mixture.

When nitrogen is present as a substrate, ammonia is produced. In

nitrogenase and hydrogenase, metal ions (iron and molybdenum) appear

to play a key role. This has been deduced from studies (Hardy and

Knight 1967; Silver 1967; Lockshin and Burris 1965; Dilworth 1966; Mor-

tenson 1966; Molar, .Burris, and Wilson 1948; Repaske and Wilson 1952;

Bulen et.al. 1965) in which ligands such as carbon monoxide, acetylene,

hydrogen, etc., inhibit the reduction of nitrogen. The presence of

paramagnetic iron and molybdenum species have been observed in the ex­

tracts by electron paramagnetic resonance spectra (Nicholas et al.

1962, Hardy et al. 1965). Analysis of the proteins obtained from the

cell-free extracts shows the existence of sulfur and inorganic sulfides

(Mortenson 1966, Tanaka et al. 1965).

"Clostridium Pasteuriamum"

"Clostridium Pasteuriamum" appears to be similar to AV except

that CP is not air-stable and different techniques must be used to pro­

duce cell-free extracts (Carnahan et al. 1960a, Carnahan et al. 1960b).

These techniques consist.of either crushing the frozen cells followed

by centrifuging or by rapid drying and extraction with 0.05 M phosphate

buffer under hydrogen and centrifuging. From the studies (the same

type as used for AV) of these extracts, of CP, their nitrogen fixation

process appears to be similar to AV; therefore it is believed that the

general mechanism of the two are similar.

19

Symbiotic

Symbiotic systems are bacteria incorporated in the root systems

of certain plants such as soybeans. There has been great difficulty in

obtaining active cell-free extracts from symbiotic systems. In 1967,

Koch, Evans; and Russell, however, did obtain an active cell-free extract

from soybean's root nodules. The investigation of these extracts has

given results similar to AV (anaerobic conditions and the requirements

of dithionite and ATP), Besides the active cell-free extracts, a non­

heme iron protein was also isolated. It has shown inconsistent results

in its ability to fix nitrogen. It has been postulated that fixation

occurs within the membrane-envelope which surrounds the bacteroids and

the leg-haemoglobin (a heme iron) (Bergersen 1960). This could explain

the inconsistent results.

ROLE OF METAL IONS IN ENZYMATIC NITROGEN FIXATION

Presence of Metals

The enzyme, nitrogenase, can be .deactivated by carbon monoxide,

hydrogen, and many other molecules which bind to metals (Hardy and

Knight 196 7; Silver 1967; Lockshin and Burris 1965; Dilworth 1966; Mor-

tenson 1966; Molar, Burris, and Wilson 1948; Repaske and Wilson 1952;

Bulen et al„ 1965). The nature of the inhibitors of nitrogenase leads

to the. concept that the metal ions in the enzyme serve as the site for

the nitrogen fixation. One of the inhibitors, acetylene, is currently

used in assaying for nitrogenase in soils (Hardy•and Knight 1967, Silver -

1967).

Several nitrogen-fixing enzymes have shown a need for both iron

and molybdenum. A kinetic study (Murray and Smith 1968, Bulen et al.

1965) has been made on the uptake of molybdenum and iron by AV. Molyb­

denum and iron were incorporated in direct proportion to the nitrogen-

fixing ability of AV. Since molybdenum doesn’t affect hydrogenase in

AV and in mycobacteria, molybdenum may serve as an electron transfer

agent in nitrogenase.

Proteins have been separated from the nitrogen-fixing sources

but their purity is still uncertain (Silver 196 7, Mortenson 1966).

Cell-free extracts from AV and CP have both been separated into two

proteins, one containing iron and molybdenum and the other containing

only iron (f erredoxin). The iron/molybdenum protein obtained from the

20

21cell-free extracts of CP has a molecular weight of 100,000 with a ratio

of iron/molybdenum/magnesium/sulfide of 12:1:1:3 (Mortenson 1966)

(another source gives the molecular weight of this protein as 100,000

with a ratio of iron/molybdenum/magnesium of 12:1:1)(Silver 1967). The

second protein is described as an iron sulfide system. The iron sulfide

protein obtained from CP (Tanaka et al. 1965) has seven non-heme iron

atoms and six or seven sulfide attached to a protein with a molecular

weight of 5800. A suggested structure for the iron sulfide system is

I I I IX S S . S S S S S\ / \ / \ / \ / \ / \ / \ /

Fe Fe Fe Fe Fe Fe Fe/ \ / \ / \ / \ / \ / \ /S S S S S S S X I I I I

The proteins obtained from the cell-free extracts of AV and CP

have been examined by electron paramagnetic resonance spectra (EPR).

Electron paramagnetic resonance spectra have shown the existence of

paramagnetic forms of iron, molybdenum, and manganese in these proteins.

In the protein obtained from AV, the EPR signals of g = 1.94, 1.97,

2.00, and 4.30 were observed (Nicholas et al. 1962). The signal, g =

1.94, was assigned to a reduced non-heme iron and the signals of g =

1.97, 2,00, and 4.30 were assigned to molybdenum (V) because on further

oxidation or reduction the signals disappeared. In crude cell-free ex ­

tracts of CP (Nicholas et al. 1962, Hardy et al. 1965), the signal of

g = 2.01 and 1.94 were fou n d . The signal g =• 2.01 was very strong and

was assigned to m a n g a nese(Il). When the cell-free extracts of CP are

purified, the signal, g = 2.01, becomes weak. In the protein obtained

22

from the cell-free extracts, the manganese signal has disappeared. The

other signal, g = 1.94, was again assigned to iron. Even though a

molybdenum signal was not observed, it cannot be concluded that there

is no molybdenum in the protein or in the cell-free extracts of CP as

the molybdenum could be in an oxidization state that is not paramagnetic,

therefore producing no EPR signal.

Mechanism and Intermediates

A general mechanism for enzymatic nitrogen fixation has been

postulated. The main points of this mechanism are (l) nitrogen is

bound to an activating site (probably an iron and/or molybdenum), (2)

the nitrogen molecule is "activated," (3) the bound nitrogen is reduced

by electron transfer and protonation yielding ammonia, and (4) the

source of the energy for the reduction is supplied by the conversion of

adenosine triphosphate (ATP) to adenosine diphosphate (ADP).

enzymatic nitrogen fixation, several more detailed mechanisms have been

proposed. Five of these detailed mechanisms will be considered here.

The first mechanism (Hardy and Knight 1966) to be considered is

a general overview of the entire biochemical process. The mechanism is

Following the general principles of the postulated mechanism for

metal thio containing electron

activating site

N

NIIIBinding

Site

ATP ADP

23

The dithionite serves as the electron donor and the ATP as the energy

source.

The second mechanism (Bulen et al. 1965) deals only with the

enzyme and the binding site for nitrogenase. The second mechanism is

Mop

Fe +"31

Fe +3

M o l

Fe^lne ]

Fe.

Mo Mo

Fe FeADPATP.

FeFe

Mo

2NHFe

6H+Fe

The third to fifth mechanisms deal only with the binding site

(the formation of the dinitrogen complex) and the reduction of the n i ­

trogen to ammonia. The third mechanism (Murray and Smith 1968) is

rM M-i .NsN.+ N, M" *M H ^

J donor [

H H I IM-N-N-M3

^ rM -N H o H N-M-. donor ______2 2 |H

donor + 2NH.

The fourth mechanism (Borod'ko and Shilov 1969) is similar to

the third except that the hydrogen is donated from the metalloenzyme

system. The fourth mechanism is

Hx /H[~M M-| +

H x /H/N-NXL?_____

H. ,NBN._ H rM'' M-|

24

The fifth mechanism is the same as the third except that the

binding site involves two metalloenzymes (four metal i o n s ) . The fifth

mechanism (Murray and Smith 1968) is

M M ‘-mT Tm-I „+ 31-̂2 f ~] + N 0 5*- ,'N=N.\ ----- H— N=N — H

pH* 'M-| j-M M-j

1 H -M M-tN H 0 H 0N — 2NH_ + 2_[_______ ]I Z Z | Je!_____

The proposed nitrogen fixation intermediates of diimide and h y ­

drazine were used in mechanisms three to five. However, no such inter­

mediates have been isolated or detected during nitrogen fixation (Mor­

ten son 1966, Bulen et al. 1965, Burris et al. 1965).

POSSIBLE ENZYME MODELS

The formation of iron complexes with molecular nitrogen is of

great interest since they may be intermediates in enzymatic nitrogen

fixation. In an attempt to prepare an enzyme model system, it was

hoped to carry out the following reactions:

00Me

v A /CO

Fe-COOC-Fe/ 'g/ \00 | 00

Me

IV

+ Na,S-C-CN

llS-C-CN

MeI

NC-C-SV .S S-C-CNii x > < ii

NC-C-S S X S-C-CN IMe

VI

NO NC-C-S I S j S-C-CN N.H.II re" X II

^S-C-CNN C - C-S^ XS /

VII Me

N N NC-C-S | .S | S-C-CN

ii X X iiNC-C-S S S-C-CN I

Me

To stabilize a molecular nitrogen complex, the metal must be electron-

rich and be able to back TT bond to the nitrogen ligand (itzkovitch and

Page 1968). Transition metal dithiolate complexes (Orgel 1966) are

easily reducible, thus making the stabilization of molecular nitrogen

feasible.

The writer, however, was unable to isolate and purify compound

VI, but the product's existence was shown by IR and analysis. To pre­

pare VI, the iron dimer, IV (King and Bisnette 1965) was reacted with V

25

26

(Davison and Holm 196 7). The reaction conditions were varied to maxi­

mize the CO evolution* Under all conditions used, CO evolution based on

the loss of four CO ligands was never complete. The following reaction

conditions were varied: the ratio of the ligand to iron (the best ratio

found was two ligands per one iron atom); different solvents (100% eth­

anol, 957o ethanol, methanol, acetone, and acetone/water); varied reac­ts otion temperature (20 to 80 ), the best temperature range appearing to

o o 'be 30 to 35 (at the higher temperatures the dimer decomposed to a

black insoluble product which exhibited no IR absorption, believed to be

an iron sulfide); and varied reaction time (general reaction time of 1

to 2 days)* After stripping the solvent and extracting the resultant

solid with pentane (compound V is insoluble), the residue was dried under

vacuum* The IR indicated the material was contaminated with compound

IV* It was impossible to separate the two compounds by fractional crys­

tallization since they have very similar solubilities. Chromatographic

separation was attempted, but the product decomposed on the column as

the dimer was eluted with pentane. The column packing utilized were

fluorosil, alumina, and cellulose*

Compound IV was also reacted with sodium dithiocarbamate. A

larger amount of gas evolved but the reaction still did not go to com­

pletion. The material obtained was soluble in ethanol, ether, pentane,

acetone, and other organic solvents * The same problems were encountered

and no solution was found.

Compound IV was also reacted with _o-phenylenebis(dimethylarsine)

(das). The reaction was run at room temperature in ethanol * The ratio

- J :

of, das to compound I was 2:1. Tlie solvent was removed by evaporation.,

and the residue extracted with pentanee The IR of the residue showed

absorption due to carbonyl and das ligands. The atom ratios found from

the elemental analysis indicated that there was one das group for every

sulfur atom. However, without further information the stoichiometry of

the complex is unknown.

PART III

CONCLUSION

28

RESUME

Since 1965 when A e D. Allen and C. V 0 Senoff discovered the

first dinitrogen complex,.many dinitrogen complexes have been prepared

and new complexes are continuously appearing in the literature. The

search for a model dinitrogen complex to serve as an intermediate, in

nitrogenase is being carried out by several inorganic chemists. The

type of dinitrogen complex to serve as a model would be a labile com­

plex which could be easily reduced and oxidized in the reactions of

converting gaseous nitrogen to ammonia (Chatt and Richards 1968; Chatt,

Nikolsky et al. 1970; Das et al. 1968). Currently no isolated dinitro­

gen complexes have been able to reduce gaseous nitrogen to ammonia.

Some unisolated dinitrogen complexes, however, have been able to reduce

gaseous nitrogen to ammonia (VoIpin and Shur 1964, Volpin and Shur

1966, Henrici-Olive and Olive 1967). These complexes might be isolated

at low temperature and/or high pressure. The search continues for a

catalytic process with a dinitrogen intermediate which will easily con­

vert nitrogen to ammonia.

29

LITERATURE CITED

Allen, A. D., and Fe Bottomley, Acc. Chern, Res., 360 (1968a) „

Allen, A. D0, and F. Bottomley, Can. J. Chem., 46, 469 (1968b).

Allen, A. D., F. Bottomley, R. 0. Harris, V e P. Reinsalu, and C. V, Senoff, J. Am. Chem. Soc., 89, 5595 (1967).

Allen, A. D., and C. V e Senoff, Chem. Comm., 621 (1965).

Allen, A. D„, and J. R. Stevens, Chem. Comm., 1147 (1967).

Andriyuk,1 K, I., Biochim. Biophys. Acta, 143, 91 (1967).

Armor, J. N., and H. Taube, J. Am. Chem. Soc., 92, 2560 (1970).

Bancroft, G. M., M. J. Mays, and B. E. Prater, Chem. Comm., 585 (1969).

Bell,- B., J. Chatt, and G. J. Leigh, Chem. Comm., 576 (1970a).

Bell, B., J. Chatt, and G. J. Leigh, Chem. Comm., 842 (1970b).

Bergersen, F. J., Bacteriol Rev., 24, 246 (i960).

Borodfko, Yu. G., and A. E. Shilov, Uspekhi Khimii, 38(5)s .355 (1969).

Bulen, W. A., J. R. LeComte, R. C. Burns, and J. Hinkson, in Non­heme Iron Proteins: Role in Energy. Conversion, A. San Pietro(Editor), Antioch Press, Ohio, 1965.

Burris, R. H., H. C. Winter, T. 0. Munson, and J. Garcia-Rivera, inNon-heme Iron Proteins: Role in Energy Conversion,. A. San Pietro (Editor), Antioch Press, Ohio, 1965.

Carnahan, J. E., L. E. Mortenson, H e F . Mower, and J. E. Castle, Bio­chim. Biophys. Acta, 38, 188 (1960a).

Carnahan, J. E., L. E. Mortenson, H. F. Mower, and J. E. Castle, Bio­chim. Biophys. Acta,,44, 520 (1960b).

Chatt, J., Chem. and Industrie, 109 (1969).

Chattj J., J. R. Dilworth, H. P. Gunz, G. J. Leigh, and J. R. Sanders,Chem. Comm., 90 (1970),

30

31

Chatty J., J» Re Dilworth, and G. J, Leigh$ Ch'em. Comm, ? 687 (1969),

Chatty J., Je R*'Dilworth, and G„ J, Leigh* J, Organomet. Chem., 21,P49 (1970).

Chatty J,* J. R. Dilworth, G. J, Leigh, and R c L, Richards,'Chem. Comm., 955 (1970).

Chatt, J„, Je R. Dilworth, R. L. Richards, and J. R. Sanders, Nature,224. 1201 (1969).

Chatt, J., and J. E. Fergusson, Chem. Comm., 126 (1968).

Chatt, J., G. J. Leigh, and R. L. Richards, Chem. Comm., 515 (1969).

'Chatt, J., D. P. Melville, and R. L. Richards, J. Chem. Soc. (A), 18,2841 (1969).

Chatt, 3., A. B. Nikolsky, R. L. Richards, J. R. Sanders, J. E. Fergus­son, and J. T. Love, J. Chem. Soc. (A), 1479 (1970).

Chatt, J., and R. L. Richards, Chem. Comm., 1522 (1968).

Coliman, J. P., and T. W. Kang, J. Am. Chem. Soc., 88, 3459 (1966).

Collman, J. P., M. Kubota, F. D. Vastine, J. Y. Sun, and J. W. Kang, J.Am. Chem. Soc., 90, 5430 (1968).

Das, P. K., J. M. Pratt, R. G. Smith, and W. J. U. Woolcock, Chem.Comm., 1539 (1968).

Davis, B.R. , N. C. Payne, and J. A. Iber.s, Inorg. Chem., _8, 2719 (1969).

Davison, A., and R. H. Holm, Inorganic Syntheses, X, 11 (1967).

Diamantis, A. A., and G. J. Sparrow, Chem. Comm., 469 (1969).

Diamantis, A. A., and G. J. Sparrow, Chem. Comm., 819 (1970).

Dilworth, M. J*, Biochim. Biophys. Acta, 127, 285 (1966).

Dilworth, M. J., D. Subramanian, T. 0. Munson, and R. H. Burris, Biochim. Biosphys. Acta, 99, 486 (1965).

Douglas, P. G. , R. D. Feltham,. and H." G. Metzger, Chem. Comm. ,889 (1970).

Enemark, J. H., B. R. Davis, J. A. McGinnety, and J. A. Ibers, Chem.Comm., 96 (1968).

32

Farquhart* E. Le 5 Le Rusnock, and Se Je Gill, Je Am. Chem. Soc., 92,416 (1970).

Fay, P*, and R. M. Cox, Biochim. Biophys. Acta, 143, 562 (1967).

Fergusson, J. E., and J. Le Love, Chem. Comm., 399 (1969).

Hardy, R. ‘W. F., and R. C. Burns, Ann. Rev. Biochem.37, 331 (1968).

Hardy, R. W; F., and E. Knight, Jr., Biochim. Biophys. Acta, 122, 520(1966).

Hardy, R. W, F., and E. Knight, Jr., Biochim. Biophys. Acta, 139, 69(1967).

Hardy, R. W. F.,"E. Knight, Jr., C. C. McDonald, and A. J. D. Eustachio, in Non-heme Iron Proteins: Role in Energy Con v e r s i o n , A. San Pietro (Editor), Antioch Press, Ohio, 1965.

Harris, R. 0., and B. A. Wright, Can. J. Chem., 48, 1815 (1970).

Harrison, D. E., and H„ Taube, J. Am. Chem. Soc., 89, 5706 (1967).

Harrison, D. E., E. Weissberger, and H. Taube, Science, 159, 320(1968).

Henrici-Olive, G . , and S. Olive, Angew. Chem., 79, 897 (1967).

Hidai, M., K. Tominari, Y. Uchida, and A. Misono, Chem. Comm., 1392(1969).

Ito, T. , S'. Kitazume, A. Yamamoto, and S. Ikeda, J. Am. Chem. Soc. , 92, 3011 (1970).

Itzkovitch, I. J., and J. A. Page, Can. J. Chem., 46, 2743 (1968).

Jolly, P. W 6 , and K. Jonas, Angew, Chem., 7_, 731 (1968).

Kane-Maguire, L. A. P., F. Basolo, and R. G. Pearson, J. Am. Chem. Soc., 91, 4609 (1969).

Kane-Maguire, L. A. P., P.'S. Sheridan, F. Basolo, and R. G. Pearson,J. Am. Chem. Soc., 90, 5295 (1968).

King, R. B. , and M. B. Bisnette, Inorg. Chem., _4, 1664 (1965).

Knoth, W. H.; J. Am. Chem. Soc., 90, 7172 (1968).

Koch, B., H. J. Evans, and S. Russell, Plant Physiol, .42, 466 (1967).

33Lockshin,•A., and H. Burris, Biochim. Biophyse Acta, 111, 1 (1965),

Lorbeth, J., He Noth, and P. V. Rinze, J6 Organomet„ Chem., 16, 1,(1969).

Misono, A., Y. Uchida, M. Hidai, and T, Ruse, Chem. Comm.9 208 (1969).

Misono, A., Y. Uchida,' and Te Saito, Bullc Chem. Soc. Japan, 40, 700 (1967).

Misono, A., Y. Uchida, T0 Saito, and K. M. Song, Chem. Comm., 419 (1967).

Mo elvTyn-Hughes, J. T., and A. W. B. Garner, Chem. Comm.,. 1309 (1969).

Molar, D. M., R. H. Burris, and P. W. Wilson, J. Am. Chem. Soc., 70,1713 (1948).

Mortenson, L, E., Biochim. Biophys. Acta, 81, 71 (1964).

Mortenson, L. E., Biochim. Biophys. Acta, 127, 18 (1966).

Murray, R. , and D. C. Smith, Coordin. Chem. Rev. , 3_, 429 (1968).

Nicholas, D. J. D., Biol. Rev., 38, 530 (1963).

Nicholas, D. J. D., D„ J. Silvester, and J. F; Fowler, Nature, 189, 634(1961).

Nicholas, D. J. D., P.. W. Wilson, W. Heinen, G. Palmer, and H. Beinert, Nature, 196, 433 (1962).

Orgel, L. E., An Introduction to Transition-metal Chemistry: Ligand- field Theory, London,. Methuene, New York, Wiley, 1966.

Parshall, G. W., J. Am. Chem. Soc., 90, 1669 (1968).

Patil, R. B., R. M. Pengra, and D. C. Yoch, Biochim. Biophys. Acta, 136 *1 (1967).

Repaske, R., and P. W. Wilson, J. Am, Chem. Soc., 74, 3101 (1952).

Rossi, M., and A, Sacco, Chem. Comm., 471 (1969).

Sacco, A., and M, Aresta, Chem. Comm., 1223 (1968).

Sacco, A., and M. Rossi, Che.. Comm., 316 (1967).

Scheidegger, H. A,, J. N. Armor, and H. Taube, J. Am. Chem. Soc., 90, 3263 (1968).

34

Schrauzcr, G. N., and G. Schlesinger, J. Am. Chem. S o c . , 9 2 , 1808(1970).

Shilov, A. E . , A. K. Shilova, and Yu. G. Borod'ko, Kinetika i Kataliz,7., 768 (1966).

Silver, W. S., Science, 1 5 7 , 100 (1967).

Srivastava, S. C . , and M. Bigorgne, J. O r g a n o m e t . C h e m . , 1 8 , 30 (1969).

Tanaka, M . , A. M. Benson, H. F. M o w e r , and K. T. Yasunobu, in Non-hemeIron Proteins: Role in Energy Conve r s i o n , A. San Pietro (Editor), Antioch Press, Ohio, 1965.

Ukhin, L. Y u . , Yu. A. Shvetsov, and M. L. Khidekel, Izv. Akad. N a u k . , S S S R . , Ser. Khim, 957 (1967).

Volpin, M. E . , and V. B. Shur, D o k l . Akad. Nauk. SSSR, 1 5 6 . 1102 (1964).

Volpin, M. E . , and V. B. Shur, Nature, 2 0 9 , 1236 (1966).

Yamamoto, A., L. S. Pu, S. Kitazume, and S. Ikeda, Chem. C o m m . , 79 (1967).

6 2 U