Carbon monoxide binding was studied in a collection of de novo heme proteins derived from combinatorial libraries of sequences designed to fold into 4-helix bundles. The design of the de novo sequences was based on the previously reported "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains are varied extensively.(1) The combinatorial mixture of amino acids included histidine and methionine, which ligate heme iron in natural proteins. However, no attempt was made to explicitly design a heme binding site. Nonetheless, as reported previously, approximately half of the binary code proteins bind heme.(2) This collection of novel heme proteins provides a unique opportunity for an unbiased assessment of the functional potentialities of heme proteins that have not been prejudiced either by explicit design or by evolutionary selection. To assess the capabilities of the de novo heme proteins to bind diatomic ligands, we measured the affinity for CO, the kinetics of CO binding and release, and the resonance Raman spectra of the CO complexes for eight de novo heme proteins from two combinatorial libraries. The CO binding affinities for all eight proteins were similar to that of myoglobin, with dissociation constants (K(d)) in the low nanomolar range. The CO association kinetics (k(on)) revealed that the heme environment in all eight of the de novo proteins is partially buried, and the resonance Raman studies indicated that the local environment around the bound CO is devoid of hydrogen-bonding groups. Overall, the CO binding properties of the de novo heme proteins span a narrow range of values near the center of the range observed for diverse families of natural heme proteins. The measured properties of the de novo heme proteins can be considered as a "default" range for CO binding in alpha-helical proteins that have neither been designed to bind heme or CO, nor subjected to genetic selections for heme or CO binding.