Active site structure in cytochrome c peroxidase and myoglobin mutants: effects of altered hydrogen bonding to the proximal histidine.

The globins and peroxidases, while performing completely different chemistry, share features of the iron heme active site: a protoporphyrin IX prosthetic group is linked to the protein by the proximal histidine residue. X-ray absorption spectroscopy provides a method to determine the local structure of iron heme active sites in proteins. Our previous studies using X-ray absorption spectroscopy revealed a significant difference in the Fe-N epsilon bond length between the peroxidases and the globins [for a review, see Powers, L. (1994) Molecular Electronics and Molecular Electronic Devices, Vol. 3, p 211 CRC Press Inc., Boca Raton, FL]. Globins typically have an Fe-N epsilon distance close to 2.1 A while the Fe-N epsilon distance in the peroxidases is closer to 1.9 A. We have proposed [Sinclair, R., Powers, L., Bumpus, J., Albo, A., & Brock, B. (1992) Biochemistry 31, 4892] that strong hydrogen bonding to the proximal histidine is responsible for the shorter bond length in the peroxidases. Here we use site-specific mutagenesis to eliminate the strong proximal hydrogen bonding in cytochrome c peroxidase and to introduce strong proximal hydrogen bonding in myoglobin. Consistent with our hypothesis, elimination of the Asp235-His175 hydrogen bond in CcP results in elongation of Fe-N epsilon from approximately 1.9 to approximately 2.1 A. Conversely, introduction of a similar strong proximal hydrogen bond in myoglobin shortens Fe-N epsilon from approximately 2.1 to approximately 1.9 A. These results correlate well with other biochemical data.