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 −295 mV exhibits a species (referred to as C red1 ) that was previously attributed to [Fe 4 S 4 ] C 1+ ( S = 1/2) weakly exchange-coupling with Ni 2+ ( S = 1) to yield apparent g -values of ( g z, y, x = 2.03, 1.88, 1.71). UV−visible absorption spectroscopy showed only one [Fe 4 S 4 ] 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. Soc. In press) to a [(CO L )Fe 3+ -Ni 2+ -H - ] 4+ cluster, a careful search for similar resonances in the EPR spectrum of the enzyme state of wild-type CODH producing C red1 was undertaken. Coupled putative [(CO L )Fe 3+ -Ni 2+ -H - ] 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 red1 . Instead of coupling with Ni 2+ ( S = 1), we propose [Fe 4 S 4 ] C 1+ ( S = 1/2) couples with [(CO L )Fe 3+ -Ni 2+ -H - ] 4+ ( S = 1/2). The putative [FeNi] signals were heterogeneous, but this heterogeneity could be removed by preincubation with CO prior to subsequent poising. We propose that an unreactive CO molecule (CO 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 S ). Either Zn 2+ or Co 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 we call C 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 4 S 4 ] 1+ clusters. These resonances are attributed in fully reduced CODH to spin−spin coupling between [Fe 4 S 4 ] C 1+ ( S = 1/2) and [Fe 4 S 4 ] B 1+ ( S = 1/2). When CODH was poised at a calculated potential of −326 mV, the UV−visible absorption spectrum indicated that only one of the [Fe 4 S 4 ] clusters was reduced. However, the EPR spectrum was much different than that observed at ca. −295 mV. The EPR spectrum of CODH at −326 mV exhibited resonances arising from a slow-relaxing [Fe 4 S 4 ] 1+ ( S = 1/2) cluster ( g z, y, x = 2.04, 1.93, 1.89) and a very minor amount of a fast-relaxing [Fe 4 S 4 ] 1+ ( S = 1/2) cluster. None of the C red1 coupling signal was present. The fast-relaxing cluster is assigned to [Fe 4 S 4 ] B 1+, while the slow-relaxing cluster is assigned to uncoupled [Fe 4 S 4 ] C 1+ . The observation of uncoupled [Fe 4 S 4 ] C 1+ at slightly lower potentials suggests the reduction of [(CO L )Fe 3+ -Ni 2+ -H - ] 4+ ( S = 1/2) to [(CO L )Fe 2+ -Ni 2+ -H - ] 3+ ( S = 0). Treatment of CODH with its physiological product (CO 2 ) 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 method of generation and the relaxation properties of the species, the g = 1.75 feature is assigned to [Fe 4 S 4 ] C 1+ ( S = 1/2) spin-coupling with [Fe 2+ -Ni 2+ ] 4+ ( S = 1) (and is referred to as C red2B ). Based on the data presented in this and Part 1, a mechanism for the oxidation of CO to CO 2 by R. rubrum CODH is proposed.