Nitrogenase structure
The reaction is carried out by an enzyme known as nitrogenase. Until recently, there was thought to be only one type of nitrogenase that was possessed by all N2-fixing organisms, and was highly conserved between species. This is the molybdenum (Mo) nitrogenase, which is indeed in almost every known N2-fixing organism. However, two alternative nitrogenase enzymes have been found in Azotobacter and other bacterial species in recent years, and these will also be discussed. In addition a totally different form of nitrogenase has reportedly been isolated fromStreptomyces thermo- autotrophicus(Ribbeet al., 1997). This enzyme is very unusual and apparently uses superoxide as its reductant and contains a molybdo-pterin cofactor active site. Since it seems to be distributed very sparsely, it will not be considered further.
The Mo nitrogenase has been most extensively studied inKlebsiella pneumoniae, A. vinelandii andClostridium pasteurianum. The enzyme consists of two compo- nents, each referred to in several ways. Component 1 contains the active site where N2 is actually reduced, and is also known as the MoFe protein or dinitrogenase.
Component 2, amongst other roles, provides electrons to Component 1 for N2
reduction and is known as the Fe protein or dinitrogenase reductase. The three- dimensional X-ray crystallographic structures of both proteins fromA. vinelandiiand C. pasteurianumand the MoFe protein fromK. pneumoniaehave been determined, as has the complex formed between the Fe protein and the MoFe protein with aluminium tetrafluoride-MgADP. The aluminium complex is believed to mimic a transition state in the hydrolysis of MgATP.
The MoFe protein is a tetramer of molecular weight around 230 kDa composed of twoasubunits encoded by thenifDgene and twobsubunits encoded by thenifK gene. The Fe protein is a homodimer of molecular weight around 60 kDa, the sub- units being encoded by thenifHgene. There is a 4Fe4S cluster in the Fe protein held symmetrically between the two subunits at one end of the interface. This protein also binds two MgATP molecules, which are thought to bind at the interface of the two subunits. The MoFe protein contains two each of two unique metal sulphur clusters.
The P clusters bind symmetrically at the interfaces of theaandbsubunits and have the stoichiometry 8Fe7S. Structurally they appear as two 4Fe4S cubes which share a sulphur atom at one corner. They are bound to the polypeptide through cysteine lig- ands to the Fe atoms with two cysteine residues, one from each subunit, each forming bridges between two iron atoms whereas the other iron atoms are bound to single cysteines. The other cluster in the MoFe protein is known as the iron molybdenum cofactor, often referred to simply as FeMoco. These cofactors are believed to be the site of N2-reduction. FeMoco has a stoichiometry of Mo7Fe8S.homocitrate and is bound to theasubunit through a cysteine residue to one iron atom and a histidine ligand to the molybdenum which also binds the homocitrate molecule. Although no other amino acid residues bind directly to FeMoco, some hydrogen bonds are formed particularly to the sulphur atoms and are apparently essential for effective activity. In the putative transition state complex between the Fe protein and the MoFe protein the structure of the Fe protein is considerably distorted. The Fe protein binds at the interface of thea andb subunits and undergoes a conformational change which
75
Z:\Customer\CABI\A4042 - Giller - Nitrogen Fixation\A4042 - Giller + Watson - Nitrogen Fixation SET #L.vp 04 June 2001 10:56:26
places the 4Fe4S cluster within 10 Å of a P cluster, which in turn is approximately 14 Å from the FeMoco centre. This juxtaposition of the metal centres in the complex is thought to indicate the electron transfer chain from the Fe protein through to FeMoco. Further detailed reviews of the structure and synthesis of nitrogenases can be found in Howard and Rees (1996) and Smith (1999).
Mechanism of N2-reduction
To reduce a molecule of N2, the nitrogenase activity begins with acquisition of electrons by the Fe protein from a strong electron donor. In all N2-fixing organisms studied, these nitrogenase-specific electron donors seem to belong to one of two groups of proteins: ferredoxins and flavodoxins. Ferredoxins belong to the class of iron–sulphur proteins discussed above, containing iron–sulphur clusters capable of transferring single electrons. Flavodoxins, in contrast, are proteins containing the prosthetic group flavin mononucleotide (FMN) and each FMN group can transfer two electrons.
So, nitrogenase activity integrates with normal cellular metabolism in part by diverting reducing equivalents – electrons – to the ferredoxin or flavodoxin that donates electrons to the Fe protein. These electrons are transferred in turn from the Fe protein to the MoFe protein. The Fe protein, in addition to the iron–sulphur cluster, has two molecules of MgATP associated with it. The reduced protein (i.e.
carrying an electron to be donated) binds to the MoFe protein. There is one binding site on each half of the the MoFe protein tetramer, and these two sites are believed to function independently. Following binding, an electron is transferred from the Fe protein to the MoFe protein with concomitant hydrolysis of both bound ATP molecules to ADP. Finally the two components of nitrogenase dissociate.
As illustrated in the equation above, in addition to the electrons whose provision we have discussed, the reaction requires an equal number of protons. Putative proton transfer pathways have been identified but the observed hydrogen evolution results
Fig. 3.1. The subunit structure of the Mo nitrogenase and the reaction of N2-reduction.
from a proportion of these electrons and protons being used ‘wastefully’ rather than being allocated to N2-reduction. H2evolution seems to be an essential part of the mechanism of N2-reduction although its precise role has yet to be determined.
Mechanisms to recover the reducing equivalents ‘lost’ in this H2 evolution are discussed below.
The final result of the reaction is, of course, reduction of N2to NH3. Because the whole reaction requires eight electrons and these are transferred one at a time from the Fe protein to the MoFe protein, it is believed that a number of enzyme- bound intermediates are formed during the reaction – rather than all eight electrons being stored and then used at once. The exact nature of the intermediates, and of the reaction, remains uncertain. The subunit structure of nitrogenase and the reaction of N2-reduction is illustrated in Fig. 3.1.
Acetylene reduction by nitrogenase
As well as the reduction of N2to NH3, nitrogenase will reduce a number of other substrates that contain triple bonds. The most important of these alternative substrates is acetylene, which is reduced to ethylene, as this reaction forms the basis of the widely used acetylene reduction assay (ARA) (Chapter 4). The reduction of acety- lene is dominant over the reduction of N2by nitrogenase, due to the higher water solubility of actylene and the higher enzyme affinity for acetylene. Thus acetylene is an effective inhibitor of N2-reduction. Hydrogen is another important inhibitor of nitrogenase, although it only inhibits reduction of N2and not of acetylene.
Alternative nitrogenases
For a long time the accepted dogma was that all nitrogenases contained Mo as a constituent of FeMoco. Since 1980 two alternative nitrogenase enzymes have been described inAzotobacterspp.: one that incorporates vanadium (V) rather than Mo, and one that has no requirement for any metal other than Fe. These are referred to as vanadium nitrogenase and the third or ‘Fe-only’ nitrogenase, respectively. There are strong similarities between the structures of each nitrogenase, but the subunits are encoded by entirely separate sets of genes –nifgenes for Mo nitrogenase,vnffor V nitrogenase andanf for the third or alternative nitrogenase. The first evidence of an alternative N2-fixation system was obtained inA. vinelandii(Bishopet al., 1980) and subsequent work has concentrated on this species, which possesses all three nitrogenase systems, onA. chroococcum, which has only the Mo and V nitrogenases, and on the Fe nitrogenase ofRhodobacter capsulatus(Eady, 1996).
Both additional nitrogenases are comprised of two component proteins with a similar subunit structure to Mo nitrogenase, except that there is an extra (d) subunit associated with the Component 1 (Eady, 1996). The stochiometry of Component 1 is thusa2b2d2. The function of thedsubunit is not known, but there is considerable sequence homology between the V nitrogenase and the third nitrogenasedsubunits,
77
Z:\Customer\CABI\A4042 - Giller - Nitrogen Fixation\A4042 - Giller + Watson - Nitrogen Fixation SET #L.vp 04 June 2001 10:56:27
so presumably they play a similar role in each. There is also significant sequence homology between the Fe protein polypeptides (encoded bynifH,vnfHandanfH, respectively) and also theaandb subunits of Component 1 (encoded bynifDK, vnfDK andanfDK) of all three enzymes. Probably the most important difference between the three nitrogenases lies in the cofactor structure, since this is the site of substrate reduction. In the V nitrogenase the cofactor contains V rather than Mo, and in the Fe nitrogenase the cofactor contains Fe (Eady, 1996).
Consistent with the idea that the cofactor forms the actual site of substrate reduction, there are marked differences in the reactions of all three nitrogenases that could be attributed in part to these different cofactors. Two differences are of importance here. One is that the reduction of N2to ammonia is less efficient in the two alternative nitrogenases, almost 50% of electrons going to production of H2, in contrast to 25% in Mo nitrogenase. The second is that both reduce acetylene (C2H2) not only to ethylene (C2H4), but also produce a small proportion (2–4%) of ethane (C2H6). Production of ethane from acetylene is very characteristic of the two alternative nitrogenases and can be detected using gas chromatography (Dilworth et al., 1987).
Neither alternative nitrogenase is normally expressed in Azotobacter in the presence of Mo (Bishopet al., 1980; Eady, 1996). In the absence of Mo, V nitrogen- ase is expressed if V is present, and the third nitrogenase is expressed only if neither metal is present. The need for additional nitrogenases remains obscure. The most obvious explanation is to enable N2-fixation to continue even in the absence of Mo (and V), but Mo-deficient soils occur only in some regions (Chapter 13), and many N2-fixing organisms have very efficient mechanisms of Mo scavenging and storage (Shahet al., 1984). Another possible rationale results from an observed difference in temperature responses for the three enzymes. While Mo nitrogenase is the most efficient at N2-reduction at 30°C, at 5°C the V nitrogenase is about six times more efficient than the Mo nitrogenase (Eady, 1996). Non-Mo nitrogenases have been identified inAzotobacterspp.,C. pasteurianum,Anabaena variabilis,R. capsulatusand Rhodospirillum rubrum. A better understanding of the role and distribution of alternative nitrogenases is required to assess their importance in nature.