A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

Abstract

BackgroundIn order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys.ResultsAmong all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme.ConclusionsWe have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.

Designed Surface Residue Substitutions in [NiFe] Hydrogenase that Improve Electron Transfer Characteristics

“…Moreover, the complex assembly pathway for this class of enzymes may also require as-yet unexplored optimizations that will require more basic study. For example, in this study and in a previous work [ 21 ], we find weak suggestion that inefficient FeS cluster insertion may be responsible for low enzyme activities. Future work on this hydrogenase and variants should focus on improving active enzyme yields to enable the observation of in vivo hydrogen production in heterologous species, especially cyanobacteria.…”

Influence of the [4Fe–4S] cluster coordinating cysteines on active site maturation and catalytic properties of C. reinhardtii [FeFe]-hydrogenase

“… 23 , 31 , 40 , 41 Exchanges of the [4Fe–4S] cluster coordinating cysteines to aspartate have been shown before to be functionally tolerated in the PsaC subunit of photosystem I, as well as in the small subunit of a [NiFe]-hydrogenase. 32 , 33 By following the occupancies of the [4Fe–4S] and [2Fe] cluster of the catalytically relevant H-cluster, we have been able to gain new insights into the importance of each individual cysteine coordinating the [4Fe–4S] cluster.…”

“…In this study the [4Fe–4S] cluster coordinating cysteines in HydA1 were individually substituted with alanine, aspartate or serine creating a pool of 12 new variants. In contrast to alanine, serine 29 – 31 and aspartate 32 , 33 may serve as direct oxygenic ligands in the form of serinate or aspartate. 23 Detailed analysis of the obtained variants by UV/vis, electron paramagnetic resonance (EPR), Fourier-transform infrared (FTIR) spectroscopy, solution based activity assays, and protein-film electrochemistry offered insights into the importance of the cysteines coordinating the [4Fe–4S] cluster.…”

From protein engineering to artificial enzymes – biological and biomimetic approaches towards sustainable hydrogen production

“…The enzyme was still an overall H 2 oxidiser, as uptake activity remained virtually unchanged at levels slightly higher than the production rate. Nevertheless, this modified hydrogenase served as a starting point for further engineering of the electron transfer chain of HynS, through replacement of all twelve coordinating amino acids by [FeS] cluster ligands found in other hydrogenases (Asp, His, Asn and Gln) 86. Albeit none of the tested mutants presented a net H 2 in vivo production, this work further showcased how tweaking the properties of the ET chain may affect the catalytic activity.…”