NH₃ (ammonia or azane gas) is an important product; it has a number of vital uses such as fertilisers, cleaners, fermentation industry and antimicrobial agent in food products. NH₃ is currently produced using the Haber-Bosch reaction technique which requires high temperature (400-500°C) and pressure (150-250 bar) which is why it is so expensive, so the production of ammonia gas in our proposed way would provide a great economic benefit.

1. Production of NH₃ in bioreactors (low-cost), instead of using the Haber-Bosch reaction technique (high-cost).
2. Scientific achievement: If our purpose succeeded, our new yeast strain, would be the first of its type; we would have an organism that "breathes" N_2, instead of O₂ for the first time.
3. Our new yeast strain would start new scientific horizons in terms of evolution, microbiology, adaptive mutations and life itself.

Photosynthesis and respiration are two vital processes that are the prerequisites for all life on Earth. The two pigments responsible for these processes are chlorophyll and heme, respectively. The so-similarity between chlorophyll and heme has been a mysterious subject of research by some researchers for a long (Vernon and Seely 1966); they are identical in structure except for the central atom: magnesium in chlorophyll and iron in heme (Bryson and Vogel 1965). Chlorophyll has been proven to have the ability to turn into haemoglobin (Hughes and Lanter 1936, Kohler et al. 1939).

O₂ is fixed by living organisms to be used as a final electron receptor, to be turned into H₂O. If we managed to replace O₂ by N₂ in yeast (because yeast contains heme) and green bacteria, we are going to obtain NH₃ and that is a real economic benefit. NH₃ (ammonia or azane gas) is an important product; it has a number of vital uses such as fertilisers, cleaners, fermentation industry and antimicrobial agent in food products. NH₃ is currently produced using the Haber-Bosch reaction technique which requires high temperature (400-500°C) and pressure (150-250 bar) which is why it is so expensive (Mike 2024), so the production of ammonia gas in our proposed way would be a great economic benefit.

Heme is the compound that forms, with some proteins, haemoglobin, the compound that exists in red blood cells in most animals. It has the ability to deliver O₂ and CO₂. Its chemical structure is C₅₅H₇₂N₄Fe. Hemocyanin is the compound that delivers O₂ and CO₂ in mollusc and arthropod species the same way hemin does, but less effectively. Its chemical structure is C₅₅H₇₂N₄Cu. Chlorophyll is the compound that fixes O₂ and CO₂ in plants. It also absorbs sun light’s energy necessary for photosynthesis. Its chemical structure is C₅₅H₇₂N₄Mg. Hemin has a semi-similarity with other compounds such as cobalamin (vitamin B₁₂) which has the Co atom as a central atom in its structure.

Accurately, gases do not bind heme; as a whole, they bind only the central atom (iron) within it. It makes a sense that, if we managed to change the central atom in heme, the fixed gas would be changed. When we have breath in our lungs, only oxygen binds haemoglobin, not because oxygen is more suitable than other gases, but because of the central atom in heme, iron, which has a high affinity with oxygen.

Iron exists naturally in the form of oxides i.e. FeO, Fe₂O₃, Fe₃O₄. Copper, in hemocyanin, exists in nature in the form of oxides too i.e. Cu₂O CuO, CuO₂, Cu₂O₃. Magnesium, in chlorophyll, also interacts with oxygen and carbon dioxide easily. Therefore, our hypothesis is "Changing the central atom in haemoglobin, hemocyanin and chlorophyll, changes the fixed gas by them".

While some researchers managed to replace the central atom in chlorophyll with copper and zinc in vitro (Petrovic et al. 2006), no attempts have been made to replace the central atom in chlorophyll and/or heme in living organisms for two reasons, according to the author’s point of view. They are:

1. The absence of purpose, which is available in this proposal; bio-production of ammonia gas.
2. Technical complexity, which may seem to be impossible at first sight for two reasons. Firstly, hemin and chlorophyll are synthesised through a set of bioreactions, so if you need to do a specific modification in heme, for instance, you will need to make a number of “specific” genetic alterations or mutations in yeast, for instance, which sounds to be impossible. Secondly, if you replaced the central atom in heme with Ca²⁺ in vitro, obtained Ca-Heme and managed to replace it with the heme of yeast (using microinjection technique), your new trait would not be inherited by next generations because of the ferrochelatase enzyme which is selective to Fe ions (Ogun et al. 2023).

Ferrochelatase (or protoporphyrin ferrochelatase) is an enzyme which is encoded by the FECH gene in humans (Anonymous In Press). It catalyses the terminal (8^th) step in the biosynthesis of heme, turning protoporphyrin IX into heme B. The interesting fact (to this proposal) is that ferrochelatase can also insert other divalent metal ions into protoporphyrin. Some ions such as Zn²⁺, Ni²⁺ and Co²⁺ form other metalloporphyrins (Medlock 2009). Zinc protoporphyrin (ZPP) is a compound found in red blood cells when heme production is inhibited by lead and/or by lack of iron (Labbe et al. 1999). Substrate inhibition happens in the order Cu²⁺ > Zn²⁺ > Co²⁺ > Ni²⁺ and can be alleviated by supplementation of metal complexing agents, such as β-mercaptoethanol or imidazole to the reaction buffer (Hunter et al. 2008). Magnesium-chelatase is a three-component enzyme that catalyses the insertion of Mg²⁺ into protoporphyrin IX. This is the first unique step in the synthesis of chlorophyll and bacteriochlorophyll (Willows 2003, Bollivar 2006).

Although it is possible to replace the central atom in heme and chlorophyll, it looks very difficult to make specific genetic manipulations to reach the target of our proposal. Therefore, our goal can be possible if we follow an adaptive mutation technique. Adaptive mutation states that, rather than mutations and evolution being random, they are responsive to certain stresses. In other words, the mutations which happen are more beneficial and specific to the given stress, neither random nor a response to anything in particular. The term stress refers to any alteration in the environment, such as temperature, nutrients, population size etc. Tests with microorganisms have found that, for adaptive mutation, more of the mutations noted after a given stress were found to be more efficient at dealing with the stress than chance alone would suggest is possible (Foster 1993, Sniegowski and Lenski 1995). Adaptive mutation is a very controversial topic so there have been many experiments to prove or discredit the theory. Three key examinations are the SOS response (McKenzie 2000), responses to starvation in Escherichia coli (Foster 2000) and testing for revertants of a tryptophan auxotroph of Saccharomyces cerevisiae (Foster 2000).

Proposed experiment

It is recommended to apply Biosafety Level 2 (BSL-2) (Anonymous 2015) during working on this experiment and to keep our (to be) new yeast strain "safe" in the laboratory. The new yeast strain must not be used for commercial purposes before carefully studying the risks and/or the advantages of its possible spread in nature. If there would be high risks associated with its possible spread, its employment for commercial purposes would not be advised.