Katsuyoshi Harada

A role of the heme-7-propionate side chain in cytochrome P450cam as a gate for regulating the access of water molecules to the substrate-binding site

Cytochrome P450cam is a heme-containing enzyme which catalyzes hydroxylation of d-camphor. The heme is bound in the heme pocket via noncovalent interactions, where two heme-propionate side chains interact with Arg, His, and/or Asp residues. To understand the role of the heme-7-propionate side chain, we prepared reconstituted P450cam with an artificial one-legged heme which has a methyl group at the position of the 7-propionate. Removal of 7-propionate dramatically decreases the d-camphor affinity by 3 orders of magnitude relative to that of the wild-type enzyme, and spectroscopic data indicate that 74% of the ferric P450cam exhibits a low-spin state owing to water molecule occupancy in the substrate-binding site under the normal assay conditions. Thus, the monooxygenase activity of the reconstituted protein is remarkably low due to the decrease in the rate of the first electron transfer from reduced putidaredoxin, whereas 87% of oxidized NADH was utilized to produce 5-hydroxy-d-camphor without any significant uncoupling reactions. X-ray structural analysis of the reconstituted enzyme reveals a novel water array extending from the substrate-binding site to bulk solvent through the position occupied by 7-propionate. This water array appears without causing any major changes in the protein structure with the notable exception of conformational changes occurring at Asp297 and Gln322 residues. We propose that the 7-propionate forms a barrier against entry of bulk water molecules and therefore in combination with Asp297, Arg299, and Gln322 plays an essential role in the process of elimination of the substrate-binding site water cluster which occurs upon d-camphor binding.

Pathway of Information Transmission from Heme to Protein upon Ligand Binding/Dissociation in Myoglobin Revealed by UV Resonance Raman Spectroscopy

Gas sensory heme proteins respond to their environment by binding a specific gas molecule to heme and transmitting this primary binding signal to the protein. How the binding signal is transmitted from the heme to the protein remains to be clarified. Using UV resonance Raman (UVRR) spectroscopy, we investigated this pathway in sperm whale myoglobin as a model gas sensory heme protein. Based on the UVRR data and the effects of deleting one of three important pathways (His-93, 6-propionate, or 7-propionate), we determined the changes in the conformation of globin that occur upon binding of CO, nitric oxide (NO), or O2 to heme and how they are transmitted from heme to globin. The UVRR results show that heme discriminates different ligands, resulting in different conformations in the globin protein. Specifically, NO induces changes in the spectrum of Trp residues in the A-helix that are significantly different from those induced by O2 or CO binding. On the other hand, binding of O2 to heme produces changes in the Tyr residues of the H-helix that are different from those induced by CO or NO binding. Furthermore, we found that cleavage of the Fe-His-93 covalent bond eliminates communication to the terminal region of the H-helix and that the 7-propionate hydrogen-bonding network is essential for transmitting the CO or NO binding signal to the N and C termini. Finally, the 6-propionate is important only for NO binding. Thus, the hydrogen-bonding network in the protein appears to be critical for intramolecular signal transduction in gas sensory heme proteins.