Previous far-UV time-resolved optical rotatory dispersion (TRORD) studies of the sub-millisecond (burst) phase of secondary structure formation in horse and tuna cytochromes c after photoreduction in denaturant suggested that the non-native His18-Fe-His33 heme ligation dominant in the unfolded horse protein facilitated this fast folding better than did the His18-Fe-His26 coordination dominant in tuna [Chen, E., Goldbeck, R.A., and Kliger, D.S. (2003) J. Phys. Chem. A 107, 8149-8155; Chen, E., Goldbeck, R.A., and Kliger, D.S. (2004) J. Am. Chem. Soc. 126, 11175-11181]. Whether His18-Fe-His33 coordination actually facilitates fast secondary structure formation or just slows folding less than His18-Fe-His26 coordination is probed by examining the double histidine mutant H26QH33N of horse heart cytochrome c. The fast folding phase is absent in H26QH33N, indicating that His18-Fe-His33 misligation does promote fast secondary structure formation, as does His18-Fe-His26 to a lesser extent. His33 may be better able to facilitate folding because it is not as constrained by hydrogen bonding interactions in the denatured state as is His26. A greater flexibility, not only because of weakened or disrupted Van der Waals interactions in the presence of guanidine hydrochloride (GuHCl) but also because of its position relative to His18, may allow His33 to ligate to the heme group more easily than His26. These results are discussed along with the results of far-UV CD and Soret and visible region MCD measurements, which were performed to probe heme ligation in H26QH33N and to understand how GuHCl affects its folding stability and cooperativity.