Abstract
Infrared-spectroscopic studies on the [NiFe]-hydrogenase of Chromatium vinosum-enriched in 15N or 13C, as well as chemical analyses, show that this enzyme contains three non-exchangeable, intrinsic, diatomic molecules as ligands to the active site, one carbon monoxide molecule and two cyanide groups. The results form an explanation for the three non-protein ligands to iron detected in the crystal structure of the Desulfovibrio gigas hydrogenase (Volbeda, A., Garcin, E., Piras, C., De Lacey, A. I., Fernandez, V. M., Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1996) J. Am. Chem. Soc. 118, 12989-12996) and for the low spin character of the lone ferrous iron ion observed with Mössbauer spectroscopy (Surerus, K. K., Chen, M., Van der Zwaan, W., Rusnak, F. M., Kolk, M. , Duin, E. C., Albracht, S. P. J., and Münck, E. (1994) Biochemistry 33, 4980-4993). The results do not support the notion, based upon studies of Desulfovibrio vulgaris [NiFe]-hydrogenase (Higuchi, Y., Yagi, T., and Noritake, Y. (1997) Structure 5, 1671-1680), that SO is a ligand to the active site. The occurrence of both cyanide and carbon monoxide as intrinsic constituents of a prosthetic group is unprecedented in biology.
Highlights
Hydrogenases catalyze the reversible splitting of dihydrogen (H2 7 2Hϩ ϩ 2eϪ) and are common in many microorganisms
As the frequency of these bands is very sensitive to the status of the active site, it was concluded that they are due to intrinsic ligands of the active site
In this paper we present spectroscopic as well as chemical evidence that the molecules observed in the FTIR spectra of [NiFe]-hydrogenases are one CO and two CNϪ groups bound to iron in the Ni-Fe active site
Summary
C. vinosum (strain DSM 185) was grown in a 700-liter batch culture as described [14]. For 15N or 13C enrichment, cells were grown in 10-liter batch cultures with 20 mM 15NH4Cl as nitrogen source or 58 mM NaH13CO3 as carbon source. Due to the limited amounts of isotope-enriched samples, it was decided to omit the TSKDEAE column step in the purification procedure. The Ultragel ACA-44 column was replaced by a Hiload 16/60 Superdex 200 one. The purity of these samples, as determined by SDS-polyacrylamide gel electrophoresis (12%) [15], was only 40 – 60%. Protein concentrations were determined according to Ref. 16, using bovine serum albumin (BSA) as standard
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