Point mutations isolated by genetic selection. A) Location of 110 nonsilent point mutations from 21 clones. Red squares indicate mutations that neutralize a positively charged residue, green xs mark mutations of all other classes, vertical lines indicate stop codons. Also indicated are the known hydrogenase structural domains and surface-accessible domains that were inferred as described in the methods. B) The observed and expected frequency of mutations by charge change class. Nonsilent mutations were grouped by the charges of both the original and mutant amino acid. Monte Carlo simulations, described in the methods, produce the expected frequency of each charge change class. As indicated by an asterisk, mutations in which a positively charged residue is replaced with a neutral are overrepresented (p-value: 1.7·10-3). Error bars are 95% confidence intervals.