Molybdenum is the only second-row transition metal required by most living organisms, and is nearly universally distributed in biology. Enzymes containing molybdenum in their active sites have long been recognized,1 and at present over 50 molybdenum-containing enzymes have been purified and biochemically characterized; a great many more gene products have been annotated as putative molybdenum-containing proteins on the basis of genomic and bioinformatic analysis.2 In certain cases, our understanding of the relationship between enzyme structure and function is such that we can speak with confidence as to the detailed nature of the reaction mechanism and, with the availability of high-resolution X-ray crystal structures, the specific means by which transition states are stabilized and reaction rate is accelerated within the friendly confines of the active site. At the same time, our understanding of the biosynthesis of the organic cofactor that accompanies molybdenum (variously called molybdopterin or pyranopterin), the manner in which molybdenum is incorporated into it, and then further modified as necessary prior to insertion into apoprotein has also (in at least some cases) become increasingly well understood.

It is now well-established that all molybdenum-containing enzymes other than nitrogenase (in which molybdenum is incorporated into a [MoFe7S9] cluster of the active site) fall into three large and mutually exclusive families, as exemplified by the enzymes xanthine oxidase, sulfite oxidase, and DMSO reductase; these enzymes represent the focus of the present account.3 The structures of the three canonical molybdenum centers in their oxidized Mo(VI) states are shown in Figure 1, along with that for the pyranopterin cofactor. The active sites of members of the xanthine oxidase family have an LMoVIOS-(OH) structure with a square-pyramidal coordination geometry. The apical ligand is a Mo=O ligand, and the equatorial plane has two sulfurs from the enedithiolate side chain of the pyranopterin cofactor, a catalytically labile Mo–OH group, and most frequently a Mo=S. Nonfunctional forms of these enzymes exist in which the equatorial Mo=S is replaced with a second Mo=O; in at least one member of the family the Mo=S is replaced by a Mo=Se, and in others it is replaced by a more complex –S–Cu–S–Cys to give a binuclear center. Members of the sulfite oxidase family have a related LMoVIO2(S–Cys) active site, again square-pyramidal with an apical Mo=O and a bidentate enedithiolate Ligand (L) in the equatorial plane but with a second equatorial Mo=O (rather than Mo–OH) and a cysteine ligand contributed by the protein (rather than a Mo=S) completing the molybdenum coordination sphere. The final family is the most diverse structurally, although all members possess two (rather than just one) equiv of the pyranopterin cofactor and have an L2MoVIY(X) trigonal prismatic coordination geometry. DMSO reductase itself has a catalytically labile Mo=O as Y and a serinate ligand as X completing the metal coordination sphere of oxidized enzyme. Other family members have cysteine (the bacterial Nap periplasmic nitrate reductases), selenocysteine (formate dehydrogenase H), –OH (arsenite oxidase), or aspartate (the NarGHI dissimilatory nitrate reductases) in place of the serine. Some enzymes have S or even Se in place of the Mo=O group. Members of the DMSO reductase family exhibit a general structural homology to members of the aldehyde:ferredoxin oxidoreductase family of tungsten-containing enzymes;4 indeed, the first pyranopterin-containing enzyme to be crystallographically characterized was the tungsten-containing aldehyde:ferredoxin oxidoreductase from Pyrococcus furiosus,5 a fact accounting for why many workers in the field prefer “pyranopterin” (or, perhaps waggishly, “tungstopterin”) to “molybdopterin”. The term pyranopterin will generally be used in the present account.




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Figure 1


Active site structures for the three families of mononuclear molybdenum enzymes. The structures shown are, from left to right, for xanthine oxidase, sulfite oxidase, and DMSO reductase. The structure of the pyranopterin cofactor common to all of these enzymes (as well as the tungsten-containing enzymes) is given at the bottom.

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The Mononuclear Molybdenum Enzymes

The induction and repression of nitrate reductase in the fungus Aspergillus nidulans.

Reduction of nitrogenous oxides by microorganisms.

Evolution of a gene. Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation

A method for the isolation of an iron-molybdenum cofactor (FeMoCo) from component I of nitrogenase is described. This method is used to isolate FeMoCo from aerobic, anaerobic, facultative, and photosynthetic nitrogen-fixing organisms. The Fe/Mo ratio in the FeMoCo from Azotobacter vinelandii and Clostridium pasteurianum is 8:1. The FeMoCo contains six atoms of acid-labile sulfide per eight Fe atoms. Crystalline component I from A. vinelandii contains 2 Mo, 33 Fe, and 27 acid-labile sulfide atoms per molecular weight of 250,000. The specific activity of FeMoCo is 425 nmol of C(2)H(4) formed/min per nmol of Mo. There is better than 98% reconstitution between FeMoCo and inactive component I in A. vinelandii mutant strain UW45. The FeMoCo yield from component I is about 90%. FeMoCo from nitrogenase component I of C. pasteurianum, Klebsiella pneumoniae, Bacillus polymyxa, and Rhodospirillum rubrum activates inactive component I in an extract from mutant strain UW45 and follows saturation kinetics. The FeMoCo in various nitrogen-fixing organisms seems to be very similar. Wild-type A. vinelandii derepressed for nitrogenase synthesis in tungsten-containing medium and K. pneumoniae mutant strain UN109 are also activated in vitro by FeMoCo. The FeMoCo is very sensitive to oxygen, but is stable even at room temperature as long as it is kept anaerobic and in N-methylformamide, the solvent used for its isolation. FeMoCo is unstable in an aqueous environment, even though it is kept strictly anaerobic. Knowledge of the structure of this cofactor should be useful for understanding the role of molybdenum at the active site of nitrogenase, role of ligands close to molybdenum in electron and proton transfer, and the catalytic mechanism of nitrogen fixation. The FeMoCo might be used as a model for synthesizing catalysts for chemical nitrogen fixation.

https://www.pnas.org/content/pnas/74/8/3249.full.pdf

Isolation of an iron-molybdenum cofactor from nitrogenase

A Common Co-Factor for Nitrate Reductase and Xanthine Dehydrogenase which also Regulates the Synthesis of Nitrate Reductase

Production of nitrate reductase may be under the control of a regulator gene with a product which can exist as an inducer or a repressor.

Regulation of nitrate reduction in Aspergillus nidulans.

Independently Segregating Genetic Loci Concerned with Nitrate Reductase Activity in Aspergillus Nidulans

Independently segregating genetic loci concerned with nitrate reductase activity in Aspergillus nidulans.

IN the course of evolution, if maximum metabolic economy is to be ensured, the level of expression of any gene becomes adjusted wherever possible so that the concentration of gene product will be sufficient, but not excessive. In the case of genes, the products of which have a regulatory role and are therefore only needed in small quantities, it may be difficult to prevent an excess of the regulator substance from being produced. Thus in the Escherichia coli lac system, although the intracellular concentration of represser is undoubtedly low1, one i gene produces sufficient represser to control several sets of structural genes2. But I shall present here evidence to indicate that the regulator gene for the nitrate assimilation system in Aspergillus nidulans is produced in near limiting quantities such that one regulator gene makes sufficient product to control one set of structural genes, but insufficient to control two.

Evidence for a near limiting intracellular concentration of a regulator substance.

GOLDBERGER1 has drawn attention to numerous reports relating both to prokaryote and eukaryote systems in which it has been shown that an enzyme may have, in addition to its own catalytic function, a regulatory role in determining the rate of its own synthesis. He has suggested the term ‘autogenous regulation’ to describe such situations. The term ‘autoregulation’ has previously been used by others2,3 to describe this general phenomenon. Autogenous regulation is, however, itself an example of a more widespread phenomenon, whereby a protein may have a regulatory function as well as a catalytic or structural role. Thus for example, Slonimski and his coworkers4,5 have suggested that in Saccharomyces cerevisiae, iso-2-cytochrome c regulates the synthesis, not of itself, but of iso-1-cytochrome c, and I have proposed6 that in Aspergillus nidulans, nitrate reductase regulates not only its own synthesis, but also the syntheses of many catabolic enzymes.

/pdf/evolutionary-significance-of-autogenous-regulation-4o1gv0c72m.pdf

D. J. Cove

Papers

A Common Co-Factor for Nitrate Reductase and Xanthine Dehydrogenase which also Regulates the Synthesis of Nitrate Reductase

Regulation of nitrate reduction in Aspergillus nidulans.

Independently segregating genetic loci concerned with nitrate reductase activity in Aspergillus nidulans.

Evidence for a near limiting intracellular concentration of a regulator substance.

Evolutionary significance of autogenous regulation.