Rhodospirillum rubrum CO-dehydrogenase. Part 2. Spectroscopic investigation and assignment of spin - Spin coupling signals

The carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum was exa mined at several potentials. The electron paramagnetic resonance (EPR) spec trum of CODH poised at approximately -295 mV exhibits a species (referred t o as C-red1) that was previously attributed to [Fe4S4](C)(1+) (S = 1/2) wea kly exchange-coupling with Ni2+ (S = 1) to yield apparent g-values of (g(z, y,x) = 2.03, 1.88, 1.71). UV-visible absorption spectroscopy showed only on e [Fe4S4] cluster to be reduced at -295 mV. Based upon our assignment of S = 1/2 resonances in indigo carmine-poised C531A CODH (see Part 1: Staples, C. R.; Heo, J.; Spangler, N. J.; Kerby, R. L.; Roberts, G. P.; Ludden, P. W . J. Am. Chem. Sec. In press) to a [(COL)Fe3+-Ni2+-H-](4+) cluster, a caref ul search for similar resonances in the EPR spectrum of the enzyme state of wild-type CODH producing C-red1 was undertaken. Coupled putative [(COL)Fe3 +-Ni2+-H-](4+) signals were observed in low intensity, which, in conjunctio n with the other assignments, prompted a reinterpretation of the redox stat e of the enzyme producing C-red1 Instead of coupling with Ni2+ (S = 1), we propose [Fe4S4]c(1+) (S = 1/2) couples with [(COL)Fe3+-Ni2+-H-](4+) (S 1/2) . Th, putative [FeNi] signals were heterogeneous, but this heterogeneity co uld be removed by preincubation with CO prior to subsequent poising. We pro pose that an unreactive CO molecule (COL) is bound to the [FeNi] cluster, p ossibly modulating the reduction potential and activating the [FeNi] cluste r for catalysis of a substrate CO molecule (COs). Either Zn2+ or Co2+ was i ncorporated 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 we call C-red2A), and an upfield wing (shoulder) near g = 2.09. That these fea tures are observed without a paramagnetic heterometal present indicates tha t they are derived solely from the [Fe4S4](1+) clusters. These resonances a re attributed in fully reduced CODH to spin-spin coupling between [Fe4S4](C )(1+) (S = 1/2) and [Fe4S4](B)(1+) (S = 1/2) When CODH was poised at a calc ulated potential of -326 mV, the UV-visible absorption spectrum indicated t hat only one of the [Fe4S4] clusters was reduced. However, the EPR spectrum was much different than that observed at ca. -295 mV. The EPR spectrum of CODH at -326mV exhibited resonances arising from a slow-relaxing [Fe4S4](1) (S = 1/2) cluster (g,,, = 2.04, 1.93, 1.89) and a very minor amount of a fast-relaxing [Fe4S4](1+) (S = 1/2) cluster. None of the C-red1 coupling si gnal was present. The fast-relaxing cluster is assigned to [Fe4S4](B)(1+), while the slow-relaxing cluster is assigned to uncoupled [Fe4S4](C)(1+). Th e observation of uncoupled [Fe4S4](C)(1+) at slightly lower potentials sugg ests the reduction of [(COL)Fe3+-Ni2+-H-](4+) (S 1/2) to [(COL)Fe2+-Ni2+-H- ](3+) (S = 0). Treatment of CODH with its physiological product (CO2) while poised at -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(x) = 1.75. This species has relaxation properties unlike C-red2A. Based upon the meth od of generation and the relaxation properties of the species, the g = 1.75 feature is assigned to [Fe4S4](C)(1+) (S = 1/2) spin-coupling with [Fe2+-N i2+](4+) (S = 1) (and is referred to as C-red2B). Based on the data present ed in this and Part 1, a mechanism for the oxidation of CO to CO2 by R. rub rum CODH is proposed.