The Contribution of Proton-Donor pKa on Reactivity Profiles of [FeFe]-hydrogenases

Abstract

The [FeFe]-hydrogenases are enzymes that catalyze the reversible activation of H2 coupled to the reduction–oxidation of electron carriers. Members of the different taxonomic groups of [FeFe]-hydrogenases display a wide range of preference, or bias, for H2 oxidation or H2 production reactions, despite sharing a common catalytic cofactor, or H-cluster. Identifying the properties that control reactivity remains an active area of investigation, and models have emerged that include diversity in the catalytic site coordination environments and compositions of electron transfer chains. The kinetics of proton-coupled electron transfer at the H-cluster might be expected to be a point of control of reactivity. To test this hypothesis, systematic changes were made to the conserved cysteine residue that functions in proton exchange with the H-cluster in the three model enzymes: CaI, CpII, and CrHydA1. CaI and CpII both employ electron transfer accessory clusters but differ in bias, whereas CrHydA1 lacks accessory clusters having only the H-cluster. Changing from cysteine to either serine (more basic) or aspartate (more acidic) modifies the sidechain pKa and thus the barrier for the proton exchange step. The reaction rates for H2 oxidation or H2 evolution were surveyed and measured for model [FeFe]-hydrogenases, and the results show that the initial proton-transfer step in [FeFe]-hydrogenase is tightly coupled to the control of reactivity; a change from cysteine to more basic serine favored H2 oxidation in all enzymes, whereas a change to more acidic aspartate caused a shift in preference toward H2 evolution. Overall, the changes in reactivity profiles were profound, spanning 105 in ratio of the H2 oxidation-to-H2 evolution rates. The fact that the change in reactivity follows a common trend implies that the effect of changing the proton-transfer residue pKa may also be framed as an effect on the scaling relationship between the H-cluster di(thiolmethyl)amine (DTMA) ligand pKa and Em values of the H-cluster. Experimental observations that support this relationship, and how it relates to catalytic function in [FeFe]-hydrogenases, are discussed.

Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis: An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase

“… 11 , 48 It is well established that bias in hydrogenases can further be controlled by, e.g., accessory FeS clusters, which govern the intramolecular electron transfer rates. 22 , 49 In parallel, a variation in bias has previously also been observed in Group A [FeFe] hydrogenase variants in which the terminal PTP residue (C299 Cp I ) was exchanged into serine or aspartate, 11 , 50 an effect attributed to changes in relative acidity of the proton donor and the [2Fe] H subsite. 50 Still, also these two latter models struggle to fully rationalize the observed reactivity of the variants.…”

“…The larger decrease of H 2 oxidation rates is in line with an impaired proton transport affecting the faster reaction more strongly than the intrinsically slower proton reduction reaction (Figure ). However, the effects on bias as well as stabilization of new H-cluster states are difficult to reconcile with an exclusive substrate (proton) transfer influence. , It is well established that bias in hydrogenases can further be controlled by, e.g., accessory FeS clusters, which govern the intramolecular electron transfer rates. , In parallel, a variation in bias has previously also been observed in Group A [FeFe] hydrogenase variants in which the terminal PTP residue (C299 Cp I ) was exchanged into serine or aspartate, , an effect attributed to changes in relative acidity of the proton donor and the [2Fe] H subsite . Still, also these two latter models struggle to fully rationalize the observed reactivity of the variants.…”

Harnessing selenocysteine to enhance microbial cell factories for hydrogen production

“…The latter plays an important role in H 2 activation. The bridgehead amine can function as a catalytic base (Fan and Hall, 2001) to the H + binding site at the distal Fe atom of [2Fe] H , providing a key interaction point with a conserved proton-transfer pathway that terminates with a Cys within H-bonding distance of the ligand (Ginovska-Pangovska et al, 2014;Duan et al, 2018;Kisgeropoulos et al, 2022). Numerous site-directed mutagenesis studies have demonstrated the importance of the surrounding protein scaffold of the H-cluster in tuning its electronic properties and catalytic activity (Winkler et al, 2013;Stripp et al, 2022).…”

Bioinspired and biomolecular catalysts for energy conversion and storage

“…An interesting report, however, demonstrated how mutations around the active-site [FeFe] hydrogenase cluster alter the calculated pendant amine pK a and the enzyme bias towards either proton reduction or dihydrogen oxidation. The researchers found that mutations that induce a positive shift in the [FeFe] cluster reduction potential yield a decrease in the bridging amine pK a (determined through calculations), which shifts activity towards hydrogen oxidation [52]. The challenge with the studies on the enzymes is that multiple properties of the active site change from a mutation.…”