BACKGROUND
the development of hydrogen as a biofuel is appealing because unlike hydrocarbon-based biofuels, the production of hydrogen involves very few chemical steps, and thus may be more efficient.
ultimately, one goal is to couple biohydrogen production to the photosynthetic apparatus to directly and efficiently capture solar energy.
hydrogen-producing redox enzymes tend to be highly oxygen sensitive  <cit> , and the first stage of photosynthesis is the production of molecular oxygen.
using a  hydrogenase with some degree of oxygen tolerance, we elected a strategy of improving the enzyme activity by investigating alternative amino acid ligation of hydrogenase fes clusters  <cit> .
the broad family of two-subunit  hydrogenases, including  hydrogenases, have many non-cysteinyl amino acid ligands for fes clusters.
an alignment of predicted  hydrogenase amino acid sequences from sequenced genomes in the ncbi genbank database revealed several ‘unusual’ amino acids in positions normally containing cysteine and known to ligate fe-s clusters involved in electron transport to the active site .
previous researchers have heterologously expressed a small subunit containing unusual fe-s ligations and have verified the presence of intact clusters of the expected 4fe4s type in spite of these unusual amino acids  <cit> , adding confidence that the amino acids predicted at these sites are not the result of sequencing errors.
we sought to use our robust alteromonas macleodii heterologous expression system  <cit>  to conduct a preliminary examination broadly surveying these and similar substitutions, both to find a better performing enzyme and to launch interest in better understanding fes clusters.
putative fes cluster ligating residues deviating from the standard pattern seen among two-subunit -hydrogenases.
each substitution is represented by multiple members; only one representative example is listed.
question marks indicate fes cluster assignments that have not been confirmed.
asterisk  indicates a substitution not investigated in this study.
the final entry represents  hydrogenases, which do feature a cysteine in lieu of a proline.
typically, experiments substituting fe-s ligation sites substitute ligating cysteines with serines, which are the most structurally homologous to cysteine  <cit> , although histidinyl ligations have also been substituted  <cit> .
however, in nature, serinyl fes cluster ligation is, to date, unknown, although there is at least one example of a threoninyl fes ligation  <cit> .
in our study, we sought to substitute these ligating cysteines to a panel of amino acid residues suspected to ligate fes clusters in hydrogenases.
therefore, we permuted all of the amino acids in the set {asp, his, asn, gln}, which are found in various subclasses of two-subunit hydrogenase , at sites containing ligating cysteines.
although glutamic acid and lysine are also found in ligating positions of two-subunit  hydrogenases, we chose to avoid these residues.
glutamic acid was avoided because of its paucity in hydrogenase alignments at conserved cysteine positions, and its structural change relative to cysteine.
lysine was not used because of its even more exaggerated structural difference.
accommodating either of these residues would likely require additional second-sphere residue substitutions to adjust the structure of the hydrogenase.
we also surmised that residue substitutions may alter the electronic landscape of the fes cluster chain in hydrogenase to allow for improved electron flow.
following the precepts of marcus theory, an ideal electron transfer chain for hydrogen production would feature a modestly energetically downhill  series of fes clusters proceeding from the distal site and leading to the active site  <cit> .
instead, most two-subunit hydrogenases feature a 3fe4s, high potential medial cluster  <cit> .
previous work has shown a modest increase in hydrogen production upon converting this hydrogenase to a 4fe4s cluster with a corresponding lower midpoint potential, and although closer, the midpoint potential is still not tuned to an optimal range  <cit> .
although we do not yet have concrete proof that the homologous substitution in the a. macleodii hydrogenase results in a 4fe4s cluster, nor do we have midpoint potential measurements, we do observe a marked increase in hydrogen production for our first generation “g1”  engineered hydrogenase in which the medial fes cluster pro and the distal fes cluster his were each substituted for cys  <cit> .
proceeding from this point, we hoped our comprehensive substitution study could serve as an exercise to screen for a further improved enzyme.
in this work, we screened the activity of  <dig> ligation variants using a crude whole cell hydrogen evolution assay.
the whole cell assay revealed two interesting facts: first, that all substitutions homologous to those observed in the pan-hydrogenase family were tolerated enough to yield measurably active enzyme.
secondly, cysteine-to-aspartic acid substitutions as a class were generally well-tolerated .
aspartate-ligated fes clusters have been observed in the natural case of pyrococcus ferredoxin; this non-canonical ligation results in a 4fe4s cluster with an aerobically labile fe atom  <cit> .
in the case of the photosystem protein psac, artificial substitution can result in the generation of a 3fe4s cluster in lieu of a 4fe4s cluster  <cit> , but one cys-asp substitution resulted in a stable 4fe4s cluster with a decreased redox potential  <cit> .
in our a. macleodii hydrogenase substitution set, further examination of  <dig> aspartic acid variants suggested that activity differences resulted from enzyme misprocessing, observed as a lack of small subunit n-terminal cleavage.
careful isolation of the enzyme alone suggested that two of these aspartic acid-substituted enzymes may be intrinsically more efficient at hydrogen evolution than the g <dig> enzyme upon which they are based.
RESULTS
hydrogenase screening assay
we constructed  <dig> variants of hydrogenase featuring substitutions from the targeted set of four amino acids in the background of our first generation “g1”  engineered hydrogenase.
the g <dig> strain has the medial fes cluster proline to cysteine substitution and the distal fes cluster histidine to cysteine substitution  <cit> .
thus, in the g <dig> starter strain, cysteine was present in all  <dig> coordinating positions, and each of the  <dig> variants substituted one of four amino acids  at one of the  <dig> positions.when these variants were tested in a crude whole cell activity assay , several trends emerged.
first, aspartic acid was almost always the most tolerated substitution.
in addition to the p285c substitution found in  hydrogenases, which is present in our chosen background sequence, all substitutions homologous to those found in known sequences were also functional .
finally, there appeared to be “privileged” ligations  wherein no substitutions or only the aspartic acid substitution resulted in detectable activity, suggesting a higher level of sensitivity to substitution at these positions.
bacterial lysate hydrogen evolution activity assay
to confirm the qualitative validity of the whole cell screening assay, we subjected the best-performing aspartic acid substitutions and one asparagine substitution to a bacterial lysate assay similar to that which we performed in our initial development of the g <dig> enzyme  <cit> .
here the results qualitatively tracked the results observed from the initial screening experimental set .
western blot analysis of the lysates , suggested that the enzyme yield roughly tracks small subunit processing efficiency.
the immature  and mature  large subunit bands appear roughly unchanged.
there are two small subunit bands, which we believe correspond to immature upper band , and mature lower band , corresponding to cleavage by the twin arginine translocon  machinery in the sequence ata/lgn .
this cleavage only occurs after the entire enzyme is properly assembled and exported via the tat system – although in the case of the a. macleodii hydrogenase, the hydrogenase likely returns to the cytoplasm  <cit> .of these two small subunit bands, the immature fraction appears to have little correlation with the activity, but qualitatively, the mature fraction strongly correlates with the observed activities .
this could be for various reasons, such as a kinetic barrier to loading the fes cluster preventing assembly, or a downstream effect such as clearance of unstable enzyme variants by proteolysis.
these results encouraged us to conduct further study on the “best of class” aspartic acid residues to separate the enzyme intrinsic activity from these cell-dependent extrinsic factors.
doubly substituted enzyme assay
using a pooled gibson isothermal assembly strategy, we generated a comprehensive library of double substitutions  drawing from a set of distal and medial cluster substitutions .
this library also contained a handful of unplanned, spontaneous triple substitutions of unresolved assembly mechanism.
no multiply-substituted construct tested yielded detectable activity, and based on our observations following western blots of other constructs, we suspect that maturation of the hydrogenase is too compromised in the multiple substitutions.
tandem purification activity assay
we subjected the g <dig> enzyme and the two best-performing variants  to a tandem purification assay to better understand the intrinsic activity of the enzyme, removed from complicating factors of the enzyme maturation process.
the tandem purification appears to remove nearly all contaminating proteins, at least as detectable by sypro ruby-stained sds-page gels, and generates well-resolved bands that appear to correspond to the large and small subunit .
after normalizing to protein content, we observed little difference in specific activity between the three variants .
western blot analysis shows  the unit composed of the processed large subunit and unprocessed small subunit species  is a dominant portion of the total enzyme for both the c258d and c295d variants suggesting an extrinsic processing or clearance defect that results from these substitutions.
sypro ruby staining shows a qualitatively similar pattern, suggesting that the observation is not an artefact of antibody binding .
a tat-signal processing defective variant  had no detectable activity  suggesting that this p-u species represents an inactive fraction of the total enzyme.
densitometric analysis  was used to quantify the processed small subunit bands, and normalization of hydrogen evolution activity to these adjusted values based on the active band suggests that c258d is approximately 4 × more active than g <dig> and c295d is approximately 2 × more active than g <dig>  western blot densities of hynl bands correlated to total protein, further suggesting the purification procedure did not discriminate between p-u and fully processed enzyme.
CONCLUSIONS
the first objective of this study was to investigate broadly whether hydrogenases tolerate the unusual amino acid residues at the ligating positions in their primary structure.
our discovery was that the a. macleodii hydrogenase is broadly tolerant of cysteine substitutions from the set {asp, his, asn, gln}; each of these residues yielded activity when substituted at some position.
although some position/residue combinations were not tolerated, all substitutions homologous to substitutions observed in the sequence databases yielded measurable hydrogen evolution activity.
our screening assay only tested the evolution direction, so it is also possible that even among those substitutions that are unmeasurably poor in our assay, certain substitutions may remain active in the uptake direction.
we further discovered that the aspartic acid substitutions were the most active among all of the tested substitutions, likely because of a minimal structural perturbation relative to cysteine  and shared electronegative quality upon ligation to an fes cluster.
examination of the lysates further suggested that the dominant reason for the loss of activity relative to the g <dig> enzyme likely results from a failure to properly process the n-terminal tat signal; further experiments may need to be conducted to properly assess this effect, possibly by tagging and separating the two dimeric species.
although all the motifs we tested were active, their lowered activities suggests that efficiency of small subunit synthesis may depend on either second-sphere residues participating in the installation of fes clusters, or alternatively, host fes cluster synthesis machinery capable of installing fes clusters around different motifs.
the second objective of this study was to discover hydrogenases that are more active in the evolution direction, possibly achieving this by modulating the electronic landscape of the fes pathway to the active site.
normalization of hydrogen evolution to only the variant of the small subunit that is likely active suggests that the g1/c258d substitution is approximately  <dig> times more active than the g <dig> enzyme, although in order to fully take advantage of this improvement, a corresponding improvement in extrinsic processing of the enzyme would need to be developed.
the isc-related ferredoxin fdx is known to be involved in the small subunit processing of the native e. coli hydrogenases  <cit> , and overexpression of the isc cluster has been used to improve yield of cyanobacterial ferredoxins  <cit> .
a similar strategy of tandem overexpression of the native e. coli isc or suf clusters, or heterologous expression of the a. macleodii suf cluster may achieve this goal.
orthogonal strategies to improve electron transport through these fes clusters may involve manipulation of second-sphere amino acids to alter the electrostatic environment.
our results may be consistent with observations that the distal fes cluster tunes hydrogen evolution activity in a ph dependent manner when the wild-type histidinyl ligation is present  <cit> .
this study represents a starting point for further study into the nature of ‘non-canonical’ amino acid substitutions that attach to fes clusters.
our expression system, with fairly minimal effort, enables isolation of highly purified hydrogenase making it a good candidate for such studies.
future studies should also consider mechanisms featuring alternative electron transfer pathways, as dye-mediated electron transfer may introduce artefactual effects, such as delivery to clusters other than the distal cluster.
finally, hydrogenases featuring other unusual fes clusters which are ligated by lysines, or an unusual, recently discovered 4fe3s cluster  <cit> , may also be a target for comprehensive substitution to {asp, his, asn, gln} in the fashion of this experiment.

 the development of hydrogen as a biofuel is appealing because unlike hydrocarbon-based biofuels, the production of hydrogen involves very few chemical steps, and thus may be more efficient. ultimately, one goal is to couple biohydrogen production to the photosynthetic apparatus to directly and efficiently capture solar energy. hydrogen-producing redox enzymes tend to be highly oxygen sensitive, and the first stage of photosynthesis is the production of molecular oxygen. using a hydrogenase with some degree of oxygen tolerance, we elected a strategy of improving the enzyme activity by investigating alternative amino acid ligation of hydrogenase fes clusters. the broad family of two-subunit hydrogenases, including hydrogenases, have many non-cysteinyl amino acid ligands for fes clusters. an alignment of predicted hydrogenase amino acid sequences from sequenced genomes in the ncbi genbank database revealed several ‘unusual’ amino acids in positions normally containing cysteine and known to ligate fe-s clusters involved in electron transport to the active site. previous researchers have heterologously expressed a small subunit containing unusual fe-s ligations and have verified the presence of intact clusters of the expected 4fe4s type in spite of these unusual amino acids, adding confidence that the amino acids predicted at these sites are not the result of sequencing errors. we sought to use our robust alteromonas macleodii heterologous expression system to conduct a preliminary examination broadly surveying these and similar substitutions, both to find a better performing enzyme and to launch interest in better understanding fes clusters. putative fes cluster ligating residues deviating from the standard pattern seen among two-subunit -hydrogenases. each substitution is represented by multiple members; only one representative example is listed. question marks indicate fes cluster assignments that have not been confirmed. asterisk indicates a substitution not investigated in this study. the final entry represents hydrogenases, which do feature a cysteine in lieu of a proline. typically, experiments substituting fe-s ligation sites substitute ligating cysteines with serines, which are the most structurally homologous to cysteine, although histidinyl ligations have also been substituted. however, in nature, serinyl fes cluster ligation is, to date, unknown, although there is at least one example of a threoninyl fes ligation. in our study, we sought to substitute these ligating cysteines to a panel of amino acid residues suspected to ligate fes clusters in hydrogenases. therefore, we permuted all of the amino acids in the set {asp, his, asn, gln}, which are found in various subclasses of two-subunit hydrogenase, at sites containing ligating cysteines. although glutamic acid and lysine are also found in ligating positions of two-subunit hydrogenases, we chose to avoid these residues. glutamic acid was avoided because of its paucity in hydrogenase alignments at conserved cysteine positions, and its structural change relative to cysteine. lysine was not used because of its even more exaggerated structural difference. accommodating either of these residues would likely require additional second-sphere residue substitutions to adjust the structure of the hydrogenase. we also surmised that residue substitutions may alter the electronic landscape of the fes cluster chain in hydrogenase to allow for improved electron flow. following the precepts of marcus theory, an ideal electron transfer chain for hydrogen production would feature a modestly energetically downhill series of fes clusters proceeding from the distal site and leading to the active site. instead, most two-subunit hydrogenases feature a 3fe4s, high potential medial cluster. previous work has shown a modest increase in hydrogen production upon converting this hydrogenase to a 4fe4s cluster with a corresponding lower midpoint potential, and although closer, the midpoint potential is still not tuned to an optimal range. although we do not yet have concrete proof that the homologous substitution in the a. macleodii hydrogenase results in a 4fe4s cluster, nor do we have midpoint potential measurements, we do observe a marked increase in hydrogen production for our first generation “g1” engineered hydrogenase in which the medial fes cluster pro and the distal fes cluster his were each substituted for cys. proceeding from this point, we hoped our comprehensive substitution study could serve as an exercise to screen for a further improved enzyme. in this work, we screened the activity of ligation variants using a crude whole cell hydrogen evolution assay. the whole cell assay revealed two interesting facts: first, that all substitutions homologous to those observed in the pan-hydrogenase family were tolerated enough to yield measurably active enzyme. secondly, cysteine-to-aspartic acid substitutions as a class were generally well-tolerated. aspartate-ligated fes clusters have been observed in the natural case of pyrococcus ferredoxin; this non-canonical ligation results in a 4fe4s cluster with an aerobically labile fe atom. in the case of the photosystem protein psac, artificial substitution can result in the generation of a 3fe4s cluster in lieu of a 4fe4s cluster, but one cys-asp substitution resulted in a stable 4fe4s cluster with a decreased redox potential. in our a. macleodii hydrogenase substitution set, further examination of aspartic acid variants suggested that activity differences resulted from enzyme misprocessing, observed as a lack of small subunit n-terminal cleavage. careful isolation of the enzyme alone suggested that two of these aspartic acid-substituted enzymes may be intrinsically more efficient at hydrogen evolution than the g enzyme upon which they are based.  hydrogenase screening assay we constructed variants of hydrogenase featuring substitutions from the targeted set of four amino acids in the background of our first generation “g1” engineered hydrogenase. the g strain has the medial fes cluster proline to cysteine substitution and the distal fes cluster histidine to cysteine substitution. thus, in the g starter strain, cysteine was present in all coordinating positions, and each of the variants substituted one of four amino acids at one of the positions.when these variants were tested in a crude whole cell activity assay, several trends emerged. first, aspartic acid was almost always the most tolerated substitution. in addition to the p285c substitution found in hydrogenases, which is present in our chosen background sequence, all substitutions homologous to those found in known sequences were also functional. finally, there appeared to be “privileged” ligations wherein no substitutions or only the aspartic acid substitution resulted in detectable activity, suggesting a higher level of sensitivity to substitution at these positions. bacterial lysate hydrogen evolution activity assay to confirm the qualitative validity of the whole cell screening assay, we subjected the best-performing aspartic acid substitutions and one asparagine substitution to a bacterial lysate assay similar to that which we performed in our initial development of the g enzyme. here the results qualitatively tracked the results observed from the initial screening experimental set. western blot analysis of the lysates, suggested that the enzyme yield roughly tracks small subunit processing efficiency. the immature and mature large subunit bands appear roughly unchanged. there are two small subunit bands, which we believe correspond to immature upper band, and mature lower band, corresponding to cleavage by the twin arginine translocon machinery in the sequence ata/lgn. this cleavage only occurs after the entire enzyme is properly assembled and exported via the tat system – although in the case of the a. macleodii hydrogenase, the hydrogenase likely returns to the cytoplasm.of these two small subunit bands, the immature fraction appears to have little correlation with the activity, but qualitatively, the mature fraction strongly correlates with the observed activities. this could be for various reasons, such as a kinetic barrier to loading the fes cluster preventing assembly, or a downstream effect such as clearance of unstable enzyme variants by proteolysis. these results encouraged us to conduct further study on the “best of class” aspartic acid residues to separate the enzyme intrinsic activity from these cell-dependent extrinsic factors. doubly substituted enzyme assay using a pooled gibson isothermal assembly strategy, we generated a comprehensive library of double substitutions drawing from a set of distal and medial cluster substitutions. this library also contained a handful of unplanned, spontaneous triple substitutions of unresolved assembly mechanism. no multiply-substituted construct tested yielded detectable activity, and based on our observations following western blots of other constructs, we suspect that maturation of the hydrogenase is too compromised in the multiple substitutions. tandem purification activity assay we subjected the g enzyme and the two best-performing variants to a tandem purification assay to better understand the intrinsic activity of the enzyme, removed from complicating factors of the enzyme maturation process. the tandem purification appears to remove nearly all contaminating proteins, at least as detectable by sypro ruby-stained sds-page gels, and generates well-resolved bands that appear to correspond to the large and small subunit. after normalizing to protein content, we observed little difference in specific activity between the three variants. western blot analysis shows the unit composed of the processed large subunit and unprocessed small subunit species is a dominant portion of the total enzyme for both the c258d and c295d variants suggesting an extrinsic processing or clearance defect that results from these substitutions. sypro ruby staining shows a qualitatively similar pattern, suggesting that the observation is not an artefact of antibody binding. a tat-signal processing defective variant had no detectable activity suggesting that this p-u species represents an inactive fraction of the total enzyme. densitometric analysis was used to quantify the processed small subunit bands, and normalization of hydrogen evolution activity to these adjusted values based on the active band suggests that c258d is approximately 4 × more active than g and c295d is approximately 2 × more active than g western blot densities of hynl bands correlated to total protein, further suggesting the purification procedure did not discriminate between p-u and fully processed enzyme.  the first objective of this study was to investigate broadly whether hydrogenases tolerate the unusual amino acid residues at the ligating positions in their primary structure. our discovery was that the a. macleodii hydrogenase is broadly tolerant of cysteine substitutions from the set {asp, his, asn, gln}; each of these residues yielded activity when substituted at some position. although some position/residue combinations were not tolerated, all substitutions homologous to substitutions observed in the sequence databases yielded measurable hydrogen evolution activity. our screening assay only tested the evolution direction, so it is also possible that even among those substitutions that are unmeasurably poor in our assay, certain substitutions may remain active in the uptake direction. we further discovered that the aspartic acid substitutions were the most active among all of the tested substitutions, likely because of a minimal structural perturbation relative to cysteine and shared electronegative quality upon ligation to an fes cluster. examination of the lysates further suggested that the dominant reason for the loss of activity relative to the g enzyme likely results from a failure to properly process the n-terminal tat signal; further experiments may need to be conducted to properly assess this effect, possibly by tagging and separating the two dimeric species. although all the motifs we tested were active, their lowered activities suggests that efficiency of small subunit synthesis may depend on either second-sphere residues participating in the installation of fes clusters, or alternatively, host fes cluster synthesis machinery capable of installing fes clusters around different motifs. the second objective of this study was to discover hydrogenases that are more active in the evolution direction, possibly achieving this by modulating the electronic landscape of the fes pathway to the active site. normalization of hydrogen evolution to only the variant of the small subunit that is likely active suggests that the g1/c258d substitution is approximately times more active than the g enzyme, although in order to fully take advantage of this improvement, a corresponding improvement in extrinsic processing of the enzyme would need to be developed. the isc-related ferredoxin fdx is known to be involved in the small subunit processing of the native e. coli hydrogenases, and overexpression of the isc cluster has been used to improve yield of cyanobacterial ferredoxins. a similar strategy of tandem overexpression of the native e. coli isc or suf clusters, or heterologous expression of the a. macleodii suf cluster may achieve this goal. orthogonal strategies to improve electron transport through these fes clusters may involve manipulation of second-sphere amino acids to alter the electrostatic environment. our results may be consistent with observations that the distal fes cluster tunes hydrogen evolution activity in a ph dependent manner when the wild-type histidinyl ligation is present. this study represents a starting point for further study into the nature of ‘non-canonical’ amino acid substitutions that attach to fes clusters. our expression system, with fairly minimal effort, enables isolation of highly purified hydrogenase making it a good candidate for such studies. future studies should also consider mechanisms featuring alternative electron transfer pathways, as dye-mediated electron transfer may introduce artefactual effects, such as delivery to clusters other than the distal cluster. finally, hydrogenases featuring other unusual fes clusters which are ligated by lysines, or an unusual, recently discovered 4fe3s cluster, may also be a target for comprehensive substitution to {asp, his, asn, gln} in the fashion of this experiment. 
we also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “g1” improved enzyme in which the medial fes cluster pro and the distal fes cluster his were each substituted for cys. using the hydrogenase from alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids at each of the 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 have comprehensively screened 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. 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. select substitutions were purified and tested, supporting the results of the screening assay. analysis of these results confirms the importance of small subunit processing. in order to understand the effects of fes cluster attachment in hydrogenase, we undertook a study to substitute all amino acid positions normally ligating the three fes clusters in the hydrogenase small subunit. normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “g1” 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.
 in order to understand the effects of fes cluster attachment in hydrogenase, we undertook a study to substitute all 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 at each of the 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” improved enzyme in which the medial fes cluster pro and the distal fes cluster his were each substituted for cys.  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.  we have comprehensively screened 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. hydrogenaseiron-sulfur clusteralteromonas macleodii deep ecotype 