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 [Fe4S4](C)(1+) (S = 1/2) weakly 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 one [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 careful 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 conjunction with the other assignments, prompted a reinterpretation of the redox state 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 could be removed by preincubation with CO prior to subsequent poising. We propose that an unreactive CO molecule (COL) is bound to the [FeNi] cluster, possibly modulating the reduction potential and activating the [FeNi] cluster for catalysis of a substrate CO molecule (COs). Either Zn2+ or Co2+ 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 [Fe4S4](1+) clusters. These resonances are 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 calculated potential of -326 mV, the UV-visible absorption spectrum indicated that 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 signal was present. The fast-relaxing cluster is assigned to [Fe4S4](B)(1+), while the slow-relaxing cluster is assigned to uncoupled [Fe4S4](C)(1+). The observation of uncoupled [Fe4S4](C)(1+) at slightly lower potentials suggests 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 -326mV 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 [Fe4S4](C)(1+) (S = 1/2) spin-coupling with [Fe2+-Ni2+](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 CO2 by R. rubrum CODH is proposed.