Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme cytochrome c(3) (cyt c(3)) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The K(d) for the single-site binding was 8.0 microM, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly (15)N-labeled and specifically [(15)N]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt c(3) could conduct redox reactions. In light of triheme cytochrome c(7), the CO-bound cyt c(3) may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H(2) cycling. Therefore, the CO-bound cyt c(3) may play a role in maintaining electron transport pathways on accumulation of toxic CO for its utilization.