15N and 1H NMR studies of Rhodospirillum rubrum cytochrome c2

Abstract

15N-Enriched cytochrome c2 was purified from Rhodospirillum rubrum that had been grown on 15NH4Cl, and the diamagnetic iron(II) form of the cytochrome was studied by 15N and 1H NMR spectroscopy. 15N resonances of the four pyrrole nitrogens, the ligand histidine nitrogens, the highly conserved tryptophan indole nitrogen, and some proline nitrogens are assigned. The resonances of the single nonligand histidine are observed only at low pH because of severe broadening produced by proton tautomerization. The resonances of exchangeable protons bonded to the nitrogens of the ligand histidine, the tryptophan, and some amide groups are also assigned. The exchange rates of the nitrogen-bound protons vary greatly: most have half-lives of less than minutes, the indolic NH of Trp-62 exchanges with a half-time of weeks, and the ligand histidine NH proton exchanges with a half-time of months. The latter observation is indicative of extreme exclusion of solvent from the area surrounding the ligand histidine and lends credence to theories implicating the degree of hydrophobicity in this region as an important factor in adjusting the midpoint potential. The dependence of the 15N and 1H NMR spectra of ferrocytochrome c2 on pH indicates neither the Trp-62 nor the ligand His side chains become deprotonated to any appreciable extent below pH 9.5. The His-18 NH remains hydrogen bonded, presumably to the Pro-19 carboxyl group, throughout the pH titrations. Because neither deprotonated nor non-hydrogen-bonded forms of His-18 are observed in spectra of the ferrocytochrome, the participation of such forms in producing a heterogeneous population having different g tensor values seems unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)