MoFe nitrogenase

The work described above was greatly aided by the provision of samples from colleagues D. gigas Ni(II)-substituted rubredoxin, I. Moura and J. J. G. Moura T. thermophilus ferredoxin, J. A. Fee VFe and MoFe nitrogenase proteins from A. vinelandii, B. J. Hales. Research in the author s laboratory is supported by grants from NIH (GM33806) and NSF (DMB8796212) and an Alfred P. Sloan Research Fellowship. 

There are several circumstantial lines of evidence that indicate that N2 binds to the enzyme by displacing H2 (i.e., that the binding site of the enzyme is a metal hydride). The main evidence for a hydride site is (1) that H2 is a specific, competitive inhibitor for the reduction of N2 (2) the limiting stoichiometry of the MoFe nitrogenase in which we observe the apparent obligatory evolution of H2 during... 

Palmer, J.G., Doemeny, P. A., and Schrauzer, G. N. (2001), The chemical evolution of a nitrogenase model, XXIII. The nature of the active site and the role ofhomocitric acid in MoFe-nitrogenase. Z. Naturforsch., B Chem. Sci. 56,386-393. 

Approximately 25% of the amino-acid residues in all gpnifHs, the Fe-protein components of MoFe nitrogenases, are conserved [271]. The amino-acid sequences of the methanogen gpnifHs, which contain from 263 to 292 residues, are themselves only... 

The a and (3 subunits of nitrogenase, encoded by the M. thermolithotrophicus niJD and niJK genes, are also predicted to contain amino-acid sequences conserved in the a and (3 subunits of the MoFe nitrogenases encoded by niJD and niJK genes in bacteria [180]. 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

Fig. 4. Requirements, substrates, and products of Mo-nitrogenase catalysis, where I is the MoFe protein II the Fe protein and Pi is inorganic phosphate. The generating system is composed of creatine phosphate and creatine phosphokinase to recycle the inhibitory MgADP produced during catalysis to...

Fig. 6. View of the nitrogenase MoFe protein P-cluster pair where ( ) represents Fe, (O) S, and (Q) C as modeled (153). The side chain of one of the...

Fig. 7. View of the FeMo-cofactor prosthetic group of the nitrogenase MoFe protein with some of the surrounding amino acid residues where ( ) represents the molybdenum coordinated to a-His-442 and homocitrate (at the top), ( ) represents the iron, interspersed with the sulfur (O) and carbon...

In late 1992 the first crystal structures of the Fe and MoFe proteins of Mo nitrogenase frora Azotobacter vinelandii were published (1-3). 

The nitrogenase proteins are generally characterized by two letters indicating the species and strains of bacteria and the numerals 1 for the MoFe protein and 2 for the Fe protein. Thus, the Fe protein from Azotobacter vinelandii is Av2 and the MoFe protein from Klebsiella pneumoniae is Kpl. 

As well as donating electrons to the MoFe protein, the Fe protein has at least two and possibly three other functions (see Section IV,C) It is involved in the biosynthesis of the iron molybdenum cofactor, FeMoco it is required for insertion of the FeMoco into the MoFe protein polypeptides and it has been implicated in the regulation of the biosynthesis of the alternative nitrogenases. 

In general there are few reproducible data on binding of reducible substrates to the isolated MoFe proteins. However, the S = EPR signal from the FeMoco centers of Kpl is pH dependent, the g values changing with a pKa of 8.7 (50). Of course, the proton is a substrate of nitrogenase however, there is no direct evidence for the proton associated with the pKa being bound directly to FeMoco. Nevertheless, this pKa can be perturbed by addition of the analog substrate acety-... 

A comprehensive description of the mechanism of molybdenum nitrogenase has been provided by the Lowe-Thorneley scheme 102) (Figs. 8 and 9). In this scheme the Fe protein (with MgATP) functions as a single electron donor to the MoFe protein in the Fe protein cycle (Fig. 8), which is broken down into four discrete steps, each of which may be a composite of several reactions ... 

Fig. 8. The Fe protein cycle of molybdenum nitrogenase. This cycle describes the transfer of one electron from the Fe protein (F) to one afi half of the MoFe protein (M) with the accompEmying hydrolysis of 2MgATP to 2MgADP + 2Pf. The rate-determining step is the dissociation of F (MgADP)2 from M,rf. Subscript red = reduced and ox = oxidized.

Fig. 9. The MoFe protein cycle of molybdenum nitrogenase. This cycle depicts a plausible sequence of events in the reduction of N2 to 2NH3 + H2. The scheme is based on well-characterized model chemistry (15, 105) and on the pre-steady-state kinetics of product formation by nitrogenase (102). The enzymic process has not been chsiracter-ized beyond M5 because the chemicals used to quench the reactions hydrolyze metal nitrides. As in Fig. 8, M represents an aji half of the MoFe protein. Subscripts 0-7 indicate the number of electrons trsmsferred to M from the Fe protein via the cycle of Fig. 8.

A further important discovery has resulted from studies on complex formation (114). With K. pneumoniae nitrogenase it has been shown that the MoFe protein containing a full complement of metal is com-... 

---