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1. Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired

   LaSarre, B; Morlen, R; Neumann, GC; Harwood, CS; McKinlay, JB (September 2023, bioRxiv)

   Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one, or some, of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2 and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used for both electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in the dark. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a β-galactosidase reporter we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2-, but not NO3-, activated N2O reduction but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use. 

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2. Tyrosine, cysteine, and proton coupled electron transfer in a ribonucleotide reductase-inspired beta hairpin maquette

   https://doi.org/10.1039/C9CC04067F  

   McCaslin, Tyler G.; Pagba, Cynthia V.; Hwang, Hyea; Gumbart, James C.; Chi, San-Hui; Perry, Joseph W.; Barry, Bridgette A. (August 2019, Chemical Communications)

   Tyrosine residues act as intermediates in proton coupled electron transfer reactions (PCET) in proteins. For example, in ribonucleotide reductase (RNR), a tyrosyl radical oxidizes an active site cysteine via a 35 Å pathway that contains multiple aromatic groups. When singlet tyrosine is oxidized, the radical becomes a strong acid, and proton transfer reactions, which are coupled with the redox reaction, may be used to control reaction rate. Here, we characterize a tyrosine-containing beta hairpin, Peptide O, which has a cross-strand, noncovalent interaction between its single tyrosine, Y5, and a cysteine (C14). Circular dichroism provides evidence for a thermostable beta-turn. EPR spectroscopy shows that Peptide O forms a neutral tyrosyl radical after UV photolysis at 160 K. Molecular dynamics simulations support a phenolic/SH interaction in the tyrosine singlet and radical states. Differential pulse voltammetry exhibits pH dependence consistent with the formation of a neutral tyrosyl radical and a p K a change in two other residues. A redox-coupled decrease in cysteine p K a from 9 (singlet) to 6.9 (radical) is assigned. At pD 11, picosecond transient absorption spectroscopy after UV photolysis monitors tyrosyl radical recombination via electron transfer (ET). The ET rate in Peptide O is indistinguishable from the ET rates observed in peptides containing a histidine and a cyclohexylalanine (Cha) at position 14. However, at pD 9, the tyrosyl radical decays via PCET, and the decay rate is slowed, when compared to the histidine 14 variant. Notably, the decay rate is accelerated, when compared to the Cha 14 variant. We conclude that redox coupling between tyrosine and cysteine can act as a PCET control mechanism in proteins. 

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3. Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired

   https://doi.org/10.1128/aem.01741-23  

   LaSarre, Breah; Morlen, Ryan; Neumann, Gina C; Harwood, Caroline S; McKinlay, James B (January 2024, Applied and Environmental Microbiology)

   Bose, Arpita (Ed.)

   ABSTRACT Denitrification is a form of anaerobic respiration wherein nitrate (NO₃⁻) is sequentially reduced via nitrite (NO₂⁻), nitric oxide, and nitrous oxide (N₂O) to dinitrogen gas (N₂) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria,Rhodopseudomonas palustrisCGA0092 andRhodobacter capsulatusSB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO₂⁻to N₂, and SB1003 reduced N₂O to N₂. For each bacterium, N₂O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N₂O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO₃⁻served as a stable, non-catalyzable molecule that was sufficient to activate N₂O reduction. Using a β-galactosidase reporter, we found that NO₃⁻acted, at least in part, by stimulating N₂O reductase gene expression. In SB1003, NO₂⁻but not NO₃⁻activated N₂O reduction, but NO₂⁻was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use. IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions. 

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4. A Copper(II)‐Nitrite Complex Hydrogen‐Bonded to a Protonated Amine in the Second‐Coordination‐Sphere

   https://doi.org/10.1002/ejic.202200105  

   Gupta, Shourya; Arora, Sumangla; Mondal, Aditesh; Stieber, S. Chantal E.; Gupta, Puneet; Kundu, Subrata (May 2022, European Journal of Inorganic Chemistry)

   Abstract Nitrous acid (HONO) plays pivotal roles in various metal‐free as well as metal‐mediated routes relevant to biogeochemistry, atmospheric chemistry, and mammalian physiology. While the metastable nature of HONO hinders the detailed investigations into its reactivity at a transition metal site, this report herein utilizes a heteroditopic copper(II) cryptate [oC]Cu^IIfeaturing a proton‐responsive second‐coordination‐sphere located at a suitable distance from a [Cu^II](ONO) core, thereby enabling isolation of a [Cu^II](κ¹‐ONO⋅⋅⋅H⁺) complex (2H‐NO₂). A set of complementary analytical studies (UV‐vis,¹⁴N/¹⁵N FTIR,¹⁵N NMR, HRMS, EPR, and CHN) on2H‐NO₂and its¹⁵N‐isotopomer (2H‐¹⁵NO₂) reveals the formulation of2H‐NO₂as {[oCH]Cu^II(κ¹‐ONO)}(ClO₄)₂. Non‐covalent interaction index (NCI) based on reduced density gradient (RDG) analysis on {[oCH]Cu^II(κ¹‐ONO)}²⁺discloses a H‐bonding interaction between the apical 3° ammonium site and the nitrite anion bound to the copper(II) site. The FTIR spectra of [Cu^II](κ¹‐ONO⋅⋅⋅H⁺) species (2H‐NO₂) shows a shift of ammonium NH vibrational feature to a lower wavenumber due to the H‐bonding interaction with nitrite. The reactivity profile of [Cu^II](κ¹‐ONO⋅⋅⋅H⁺) species (2H‐NO₂) towards anaerobic nitration of substituted phenol (2,4‐DTBP) is distinctly different relative to that of the closely related tripodal [Cu^II]‐nitrite complexes (1‐NO₂/3‐NO₂/4‐NO₂). 

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5. Buffer Assists Electrocatalytic Nitrite Reduction by a Cobalt Macrocycle Complex

   https://doi.org/10.1021/acs.inorgchem.2c00909  

   Braley, Sarah E.; Kwon, Hyuk-Yong; Xu, Song; Dalton, Evan Z.; Jakubikova, Elena; Smith, Jeremy M. (August 2022, Inorganic Chemistry)

   This work reports a combined experimental and computational study of the activation of an otherwise catalytically inactive cobalt complex, [Co(TIM)Br2]+, for aqueous nitrite reduction. The presence of phosphate buffer leads to efficient electrocatalysis, with rapid reduction to ammonium occurring close to the thermodynamic potential and with high Faradaic efficiency. At neutral pH, increasing buffer concentrations increase catalytic current while simultaneously decreasing overpotential, although high concentrations have an inhibitory effect. Controlled potential electrolysis and rotating ring-disk electrode experiments indicate that ammonium is directly produced from nitrite by [Co(TIM)Br2]+, along with hydroxylamine. Mechanistic investigations implicate a vital role for the phosphate buffer, specifically as a proton shuttle, although high buffer concentrations inhibit catalysis. These results indicate a role for buffer in the design of electrocatalysts for nitrogen oxide conversion. 

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