SUBSTITUTION OF VALINE FOR HISTIDINE-265 IN CARBON-MONOXIDE DEHYDROGENASE FROM RHODOSPIRILLUM-RUBRUM AFFECTS ACTIVITY AND SPECTROSCOPIC STATES

In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, hi stidine 265 was replaced with valine by site-directed mutagenesis of t he cooS gene, The altered form of CODH (H265V) had a low nickel conten t and a dramatically reduced level of catalytic activity, Although tre atment with NiCl2 and CoCl2 increased the activity of H265V CODH by se veralfold, activity levels remained more than 1000-fold lower than tha t of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters, The K-m and K-D for CO as well a s the K-D for cyanide were relatively unchanged as a result of the ami no acid substitution in CODH. The time-dependent reduction of the [Fe4 S4](2+) clusters by CO occurred on a time scale of hours, suggesting t hat, as a consequence of the mutation, a rate-limiting step had been i ntroduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the g(av) = 1.86 an d g(av) = 1.87 signals characteristic of reduced forms of the active s ite (center C) of wild-type CODH, This indicates that the electronic p roperties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore.