Rhodospirillum rubrum CO-dehydrogenase. Part 2: Spectroscopic investigation and assignment of spin-spin coupling signals
The carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum was examined at several potentials. The electron paramagnetic resonance (EPR) spectrum of CODH poised at approximately {minus}295 mV exhibits a species (referred to as C{sub red1}) that was previously attributed to [Fe{sub 4}S{sub 4}]{sub C}{sup 1+} (S = 1/2) weakly exchange-coupling with Ni{sup 2+} (S = 1) to yield apparent g-values of (G{sub z,y,x} = 2.03, 1.88, 1.71). UV-visible absorption spectroscopy showed only one [Fe{sub 4}S{sub 4}] cluster to be reduced at {minus}295 mV. Based upon the assignment of S = 1/2 resonances in indigo carmine-poised C531A CODH to a [(CO{sub L})Fe{sup 3+}-Ni{sup 2+}-H{minus}]{sup 4+} cluster, a careful search for similar resonances in the EPR spectrum of the enzyme state of wild-type CODH producing C{sub red1} was undertaken. Coupled putative [(CO{sub L})Fe{sup 3+}-Ni{sup 2+}-H{minus}]{sup 4+} signals were observed in low intensity, which, in conjunction with the other assignments, prompted a reinterpretation of the redox state of the enzyme producing C{sub red1}. instead of coupling with Ni{sup 2+} (S = 1), the authors propose [Fe{sub 4}S{sub 4}]{sub C}{sup 1+} (S = 1/2) couples with [(CO{sub L})-Fe{sup 3+}-Ni{sup 2+}-H{minus}]{sup 4+} (S = 1/2). The putative [FeNi] signals were heterogeneous, but this heterogeneity could be removed by preincubation with CO prior to subsequent poising. They propose that an unreactive CO molecule (CO{sub L}) is bound to the [FeNi] cluster, possibly modulating the reduction potential and activating the [FeNi] cluster for catalysis of a substrate CO molecule (CO{sub S}). Either Zn{sup 2+} or Co{sup 2+} was incorporated into purified, Ni-deficient CODH. The EPR spectra of reduced Zn-CODH and Co-CODH contain resonances in the g = 1.73--1.76 region (which they call C{sub red2A}), and an upfield wing (shoulder) near g = 2.09. That these features are observed without a paramagnetic heterometal present indicates that they are derived solely from the [Fe{sub 4}S{sub 4}]{sup 1+} clusters. These resonances are attributed in fully reduced CODH to spin-spin coupling between [Fe{sub 4}S{sub 4}]{sub C}{sup 1+} (S = 1/2) and [Fe{sub 4}S{sub 4}]{sub B}{sup 1+} (S = 1/2). When CODH was poised at a calculated potential of {minus}326 mV, the UV-visible absorption spectrum indicated that only one of the [Fe{sub 4}S{sub 4}] clusters was reduced. However, the EPR spectrum was much different than that observed at ca. {minus}295 mV. The EPR spectrum of CODH at {minus}326 mV exhibited resonances arising from a slow-relaxing [Fe{sub 4}S{sub 4}]{sup 1+} (S = 1/2) cluster (g{sub z,y,x} = 2.04, 1.93, 1.89) and a very minor amount of a fast-relaxing [Fe{sub 4}S{sub 4}]{sup 1+} (S = 1/2) cluster. None of the C{sub red1} coupling signal was present. The fast-relaxing cluster is assigned to [Fe{sub 4}S{sub 4}]{sub C}{sup 1+}, while the slow-relaxing cluster is assigned to uncoupled [Fe{sub 4}S{sub 4}]{sub C}{sup 1+}. The observation of uncoupled [Fe{sub 4}S{sub 4}]{sub C}{sup 1+} at slightly lower potentials suggests the reduction of [(CO{sub L})Fe{sup 3+}-Ni{sup 2+}-H{minus}]{sup 4+} (S = 1/2) to [(CO{sub L})Fe{sup 2+}-Ni{sup 2+}-H{minus}]{sup 3+} (S = 0). Treatment of CODH with its physiological product (CO{sub 2}) while poised at {minus}326 mV with 99% reduced phenosafranin results in accumulation of oxidized dye, the production of CO, and the appearance of a new species with g{sub x} = 1.75. This species has relaxation properties unlike C{sub red2A}. Based upon the method of generation and the relaxation properties of the species, the g = 1.75 feature is assigned to [Fe{sub 4}S{sub 4}]{sub C}{sup 1+} (S = 1/2) spin-coupling with [Fe{sup 2+}-Ni{sup 2+}]{sup 4+} (S = 1) (and is referred to as C{sub red2B}). Based on the data presented in this and Part 1, a mechanism for the oxidation of CO to CO{sub 2} by R.rubrum CODH is proposed.
- Research Organization:
- Univ. of Wisconsin, Madison, WI (US)
- Sponsoring Organization:
- USDOE; National Institutes of Health (NIH)
- DOE Contract Number:
- FG02-87ER13691
- OSTI ID:
- 20014098
- Journal Information:
- Journal of the American Chemical Society, Vol. 121, Issue 48; Other Information: PBD: 8 Dec 1999; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
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