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

Abstract

Background: In 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.

Results: Among 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.

Conclusions: We 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.

Figure 1

Crude whole cell screening assay for hydrogen evolution activity. Relative activities of amino acid substitutions at each of the 12 substitution positions predicted to ligate Fe-S clusters are presented. Error bars represent geometric standard errors propagated through activity normalization.

Figure 2

Bacterial lysate activity assay. A)Hydrogen evolution activities of hydrogenases bearing select substitutions as ascertained by by bacterial lysate assay. Error bars represent the standard error of the mean activity. The dotted line separates Asp substitution data from Asn substitution data. B) SYPRO Ruby stain loading control for C) anti-HynL and D) anti-Strep western blots of bacterial lysates of select substitutions demonstrating approximate enzyme yield.

Figure 3

Purified protein activity assay. Left: Hydrogen evolution activities of G1, C258D, and C295D after purification using IMAC/strep-tactin tandem purification. Error bars represent the standard error of the mean activity. Right: anti-HynL and anti-strep western blots of enzyme samples.