non-competitive inhibitor, and carbon disulfide functions as a rapid-equilibrium inhibitor of nitrogenase. Vanadium nitrogenases have also been shown to catalyze the conversion of CO into alkanes through a reaction comparable to Fischer-Tropsch synthesis. Organisms that synthesize nitrogenase There are two types of bacteria that synthesize nitrogenase and are required for nitrogen fixation. These are: Free-living bacteria (non-symbiotic), examples include: Cyanobacteria (blue-green algae) Green sulfur bacteria Azotobacter Mutualistic bacteria (symbiotic), examples include: Rhizobium, associated with leguminous plants Spirillum, associated with cereal grasses Frankia Similarity to other proteins The three subunits of nitrogenase exhibit significant sequence similarity to three subunits of the light-independent version of protochlorophyllide reductase that performs the conversion of protochlorophyllide to chlorophyll. This protein is present in gymnosperms, algae, and photosynthetic bacteria but has been lost by angiosperms during evolution. Separately, two of the nitrogenase subunits (NifD and NifH) have homologues in methanogens that do not fix nitrogen e.g. Methanocaldococcus jannaschii. Little is understood about the function of these "class IV" nif genes, though they occur in many methanogens. In M. jannaschii they are known to interact with each other and are constitutively expressed. Measurement of nitrogenase activity As with many assays for enzyme activity, it is possible to estimate nitrogenase activity by measuring the rate of conversion of the substrate (N2) to the product (NH3). Since NH3 is involved in other reactions in the cell, it is often desirable to label the substrate with 15N to provide accounting or "mass balance" of the added substrate. A more common assay, the acetylene reduction assay or ARA, estimates the activity of nitrogenase by taking advantage of the ability of the enzyme to reduce acetylene gas to ethylene gas. These gases are easily quantified using gas chromatography. Though first used in a laboratory setting to measure nitrogenase activity in extracts of Clostridium pasteurianum cells, ARA has been applied to a wide range of test systems, including field studies where other techniques are difficult to deploy. For example, ARA was used successfully to demonstrate that bacteria associated with rice roots undergo seasonal and diurnal rhythms in nitrogenase activity, which were apparently controlled by the plant. Unfortunately, the conversion of data from nitrogenase assays to actual moles of N2 reduced (particularly in the case of ARA), is not always straightforward and may either underestimate or overestimate the true rate for a variety of reasons. For example, H2 competes with N2 but not acetylene