Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase

Abstract

Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in “green” energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

“…At the enzyme level, previous insights into nitrogenase sequence-function relationships have primarily derived from single or dual substitution studies. These have often yielded diminished or abolished nitrogenase activity (Seefeldt et al, 2020; Stripp et al, 2022), though in certain cases improved reactivity toward alternate, industrially relevant substrates (Fixen et al, 2016; Seefeldt et al, 2020). Despite illuminating key features of extant nitrogenase mechanism in select model organisms, the combination of detailed functional studies within an explicit evolutionary scheme has not previously been accomplished for the nitrogen fixation system.…”

“…How nitrogen fixation emerged and co-evolved under past environmental conditions is still poorly constrained relative to its importance in Earth’s planetary and biological history. For example, how the structural domains and regulatory network of nitrogenase were recruited (Boyd, Costas, Hamilton, Mus, & Peters, 2015; Mus, Colman, Peters, & Boyd, 2019), under what selective pressures the metal dependence of nitrogenases were shaped (Boyd, Hamilton, & Peters, 2011; Garcia et al, 2020), and how the interaction between peptide and metallocluster has been finely tuned to catalyze one of the most difficult reactions in nature (Harris et al, 2019; MacKay & Fryzuk, 2004; Seefeldt et al, 2020; Stripp et al, 2022), all remain open questions. In addition to revealing fundamentally important aspects of metabolic evolution, answering these questions would illuminate the engineering potential of nitrogenase as a whole.…”

Enzymatic Fischer–Tropsch-Type Reactions

“…The activity of the V-nitrogenase-expressing culture to perform CO reduction could be further improved by intermittent aeration (for 20 min) between repeated intervals (4 h each) of incubation of 15% CO with the V-nitrogenase expressing culture of A. vinelandii, an effective measure to alleviate the cells from the inhibitory effect of CO on the respiratory chain and other key metabolic pathways, yielding a TON of 7500 after 20 repetitions of this procedure. 18 Importantly, while GC-MS experiments with 13 CO confirmed the origin of C in the hydrocarbon products as that from CO, nanoscale secondary ion MS analysis revealed identical 13 C/ 12 C ratios of the V-nitrogenase expressing cultures incubated with 13 CO and 12 CO. Moreover, a comparative LC-MS analysis of cultures incubated without CO and with 13 CO or 12 CO showed no incorporation of the 13 C label into acetyl-CoA, the central metabolite, during cell growth.…”

“…18 Importantly, while GC-MS experiments with 13 CO confirmed the origin of C in the hydrocarbon products as that from CO, nanoscale secondary ion MS analysis revealed identical 13 C/ 12 C ratios of the V-nitrogenase expressing cultures incubated with 13 CO and 12 CO. Moreover, a comparative LC-MS analysis of cultures incubated without CO and with 13 CO or 12 CO showed no incorporation of the 13 C label into acetyl-CoA, the central metabolite, during cell growth. 18 These observations collectively point to the whole-cell reduction of CO by nitrogenase as a secondary metabolic pathway with potential evolutionary relevance (see section 4 for detailed discussion) while suggesting a biotechnological adaptability of this process for whole-cell production of hydrocarbons from CO reduction.…”

“…Interestingly, despite their ability to interact with CO 2 and reduce it to CO, the two Fe proteins from A. vinelandii were unable to generate hydrocarbons as products of CO 2 reduction, nor were they able to reduce CO into hydrocarbons; instead, both Fe proteins were capable of oxidizing CO to CO 2 in the presence of IDS at a considerably higher rate than that of the reduction of CO 2 to CO . The ability to enable an ambient interconversion between CO 2 and CO makes the Fe protein a functional mimic of the Ni-containing CO-dehydrogenase, with the [Fe 4 S 4 ] center of the former representing a simplified analogue of the [NiFe 4 S 4 ] cluster (or C-cluster) of the latter. − , …”