Electrocatalytic reduction and oxidation of nitrite using cobalt phthalocyanine derivatives was studied. The detection limit of 1 x 10⁻¹° mol dm⁻³ was achieved when these molecules were employed as catalysts for nitrite detection. The mechanisms for nitrite catalysis were proposed. The position of the peripheral substituents on cobalt porphyrazines (related to cobalt phthalocyanines) affected the catalytic activity of these complexes. The highest activity for nitrite reduction was observed on the cobalt(II) 2,3-tetramethyltetrapyridinoporphyrazine ([CoTm-2,3-tppa]⁴⁺), with cobalt phthalocyanine showing the lowest activity, and the cobalt(II) 3,4- tetramethyltetrapyridinoporphyrazine ([CoTm-3,4-tppa]⁴⁺), showing intermediate behaviour. A mixture of a negatively charged cobalt(II) tetrasulfophthalocyanine ([Co¹¹TSPc]⁴⁻) and a positively charged [CoTm-3,4-tppa]⁴⁺ showed better activity for nitrite reduction than did the individual components. Cobalt porphyrazines lowered the potentials for nitrite reduction in that peaking was observed, as opposed to cobalt phthalocyanine, where only the increase in currents was observed without peaking. Using the cobalt phthalocyanine derivatives, nitrite can be reduced to ammonia with high current efficiency. A glassy carbon electrode modified with [Co¹¹TSPc]⁴⁻ was employed for the determination of nitrite. Nitrate had an insignificant effect on nitrite oxidation on these modified electrodes. Electrocatalytic determination of S0₂ was studied as a function of pH at a glassy carbon electrode modified with iron(II) tetrasulfophthalocaynine. It was found that depending on pH, S0₂.xH₂0, HS0₃⁻ and/or SO₃²⁻ are the main compounds in solution and that these compounds behave differently at the electrode surface. Detection limits ranging from 4.0 ± 0.1 x 10⁻⁵ to 7.5 ± 0.1 x 10⁻⁵ mol dm⁻³ depending on pH were observed. Similar results were obtained when cobalt(II) tetrasulfophthalocaynine was employed for S0₂ catalysis under the same experimental conditions. Cysteine and histidine determination using oxidation currents was performed on glassy carbon electrodes modified with [CoTm-3,4-tppa]⁴⁺ (represented as [CoTm-3,4-tppa]⁴⁺-GCE) in pH 7 Tris buffer. The detection limit of 1.0 x 10⁻⁵ mol dm⁻³ for cysteine and 2.24 x 10⁻⁷ mol dm⁻³ for histidine were obtained. Cyanide can be detected down to 1 x 10⁻¹¹ mol dm⁻³ using [CoTm-3,4-tppa]⁴⁺-GCE in pH 10.8 buffer. Cyanide and S0₂ coordinate to the [CoTSPc]⁴⁻ species. The coordination is accompanied by oxidation of the central Co(II) metal, forming a [Co¹¹¹CoTSPc]³⁻ species. The rate constants for cyanide coordination to the [Co¹¹TSPc]⁴⁻ complex are larger than those reported for the coordination of cyanide to FePc and RuPc complexes in non-aqueous media. Autoreduction of [Co¹¹Tmtppa]⁴⁺ occurred in the presence of either histidine or cysteine, with the formation of metal reduced species, [Co¹Tmtppa(-2)]³⁺. Nitric oxide and nitrite coordinate to the [Co¹¹Tmtppa]⁴⁺ species, without auto-reduction of this species, which was observed for cysteine or histidine. The use of [Co¹¹TSPc]⁴ resulted in improved rate of interaction with nitrite when compared to the [Co¹¹Tmtppa]⁴⁺ species.

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Cyanide was rapidly removed when added to culture supernatants of seven different Pseudomonas. The ability to remove cyanide was correlated with the accumulation of α-keto acids (pyruvate and α-ketoglutarate). These compounds react with cyanide forming less toxic cyanohydrins, thus conferring a mechanism for bacterial cyanide tolerance. When added to growth media the α-keto acids were shown also to serve as effective cyanide antagonists. While all bacteria tested accumulated α-keto acids, only those capable of utilizing cyanide as a nutritional nitrogen source were able to metabolize cyanohydrins. In P. fluorescens NCIMB 11764, the same enzyme (cyanide oxygenase) shown previously to be involved in cyanide metabolism appears responsible for cyanohydrin transformation. Keto acid excretion is believed to represent a new mechanism of bacterial cyanide detoxification with further enzymatic metabolism of the cyanohydrins helping to explain how cyanide can satisfy the nitrogen requirement in cyanide-utilizing bacteria.

Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.

Ten bacteria capable of growth on the metal-cyano complex, tetracyanonickelate (II) {K2 [Ni(CN)J } (TCN), supplied as the sole nitrogen source, were isolated. Seven isolates were identified as pseudomonads while the remaining three were classified as Klebsiella species. In addition to TCN, all isolates were able to utilize KCN although it was significantly more toxic. The degradation of TCN was most complete when supplied at growth-limiting concentrations, did not occur when ammonia was present, and resulted in the formation of nickel cyanide [Ni(CN)2] as a degradation product.

Pseudomonas fluorescens NCIMB 11764 was capable of utilizing cyanide as a sole nitrogen source for growth. Cyanate (OCN") and S-cyanoalanine could also serve as nitrogenous substrates, but do not appear to play a role as intermediates in cyanide metabolism. Growth of this strain on cyanate as the sole nitrogen source led to the induction of an enzyme characterized as a cyanase (EC 3.5.5.3) based on its stoichiometric conversion of cyanate to ammonia, and dependence on bicarbonate for maximal activity. However, since cyanase activity was not elevated in cyanide-grown cells it was concluded that it serves no role in cyanide metabolism. Related studies aimed at examining a possible role for S-cyanoalanine as a cyanide-assimilation intermediate showed that while this compound also serves as a nitrogen source, it also is not important in cyanide metabolism. Studies focused on the utilization of free cyanide as a growth substrate led to the development of a fed-batch cultivation procedure greatly facilitating further experimentation aimed at the identification of cyanide metabolites. In addition to CO_2 and NH_3 as described earlier, two additional metabolites including formamide and formate were detected by using nC-NMR, HPLC, radioisotrapping methods and other analytical means. The formation of metabolites was shown to be induced after growth on cyanide with the relative product yields dependent on the availability of oxygen. These findings support earlier work in which an oxygen-dependent mechanism was proposed for the formation of C02 and NH3. However, at least two additional oxygen-independent pathways of cyanide conversion can be elaborated by this organism. One of these involves conversion to formate and ammonia while the other leads to the formation of formamide, which is not further degraded. Thus, growth on cyanide appears to occur by several mechanisms of chemical transformation presumably serving both detoxification and nutritional roles. Since two of these mechanisms generate ammonia, which is readily assimilated, growth is presumed to proceed via ammonia as a provisionary nitrogenous substrate.

One of the challenges in biology is placing a function on the myriad of gene sequences having become available from rapid advances in genome sequencing. One such example is a gene cluster (Nit1C) found in bacteria that is tied to the unusual ability of certain bacteria to grow when supplied cyanide as the sole nitrogen source. The term cyanotrophs has been applied to such bacteria, for which a genetic linkage between cyanotrophy and Nit1C was demonstrated for 10 separate bacteria. In addition to growth, cyanide induced the expression of Nit1C genes in all organisms tested, and in one case, deletion of one of the Nit1C genes (nitC) caused a loss of growth. Of the ten bacteria able to grow cyanotrophically, all gave evidence of harboring Nit1C on their genome except for two (Pseudomonas fluorescens Pf11764 and P. monteilii BCN3), which were sequenced and the presence of Nit1C was also confirmed. A broader search of bacteria identified 270 separate strains with the cluster, all limited to bacteria spanning the phyla Firmicutes, Actinobacteria, Proteobacteria and Cyanobacteria. Remarkably, many examples of a single representative of a given taxon contained Nit1C, most poignantly displayed by Pf11764 and PmBCN3; the interpretation being the cluster was likely acquired by horizontal gene transfer in response to cyanide as an environmental cue. Consistent with its absence in Archaea is the time line for the emergence of cyanide producing organisms (cyanogens) on earth dating back only 400-500 million years.

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