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 , histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl 2...

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Bibliographic Details
Published in:The Journal of biological chemistry 1998-02, Vol.273 (7), p.4059-4064
Main Authors: Spangler, N J, Meyers, M R, Gierke, K L, Kerby, R L, Roberts, G P, Ludden, P W
Format: Article
Language:English
Subjects:
Aldehyde Oxidoreductases - chemistry
Aldehyde Oxidoreductases - metabolism
Binding Sites
Carbon Monoxide - metabolism
Cobalt - pharmacology
Cyanides - metabolism
Electron Spin Resonance Spectroscopy
Iron - analysis
Iron-Sulfur Proteins - genetics
Iron-Sulfur Proteins - metabolism
Multienzyme Complexes - chemistry
Multienzyme Complexes - metabolism
Mutagenesis, Site-Directed - genetics
Nickel - analysis
Nickel - pharmacology
Rhodospirillum rubrum - enzymology
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Summary:In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum , histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl 2 and CoCl 2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe 4 S 4 clusters. The K m and K D for CO as well as the K D for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe 4 S 4 ] 2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced 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 and g av = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe 4 S 4 chromophore.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.273.7.4059