An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5

Previous Mossbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]²⁺ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel Mossbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using ¹³C-labeled CO and enzyme isotopically enriched in ⁵⁷Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a ¹³C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The ¹³C hyperfine coupling constant (A(C) ~ 21 MHz) is within the range observed for inorganic iron-carbonyl clusters. The observation of ⁵⁷Fe ENDOR signals from two types of iron site (| A(1C) | ~ 30-34 MHz; | A(2C) | ~ 6 MHz) and resolved ⁵⁷Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by | A(1C) | confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the ⁵⁷Fe hyperfine coupling constants.