Three-dimensional structure of the reduced C77S mutant of the Chromatium vinosum high-potential iron-sulfur protein through nuclear magnetic resonance: comparison with the solution structure of the wild-type protein.

The full 1H NMR assignment of the reduced C77S mutant of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) was achieved by taking advantage of the assignment available for the wild-type protein. A total of 1565 nuclear Overhauser effect (NOE) spectroscopy cross peaks were integrated and converted into distance constraints, of which 497 were found to be irrelevant. An additional 24 dipolar constraints were obtained from one-dimensional NOE difference spectra by saturating hyperfine-shifted beta CH2 cysteine/serine protons. Forty-six 3JNH-H alpha coupling constants and eight hydrogen bonds provided further constraints. Through a distance geometry approach, a family of 15 structures was calculated, which was subsequently subjected to restrained energy minimization. The root mean square deviations of the minimized structures were 0.62 +/- 0.09 and 1.09 +/- 0.11 A for backbone and heavy atoms, respectively. The resulting solution structures are very similar to those of the reduced wild-type protein (WT). An analysis of the NOEs experienced by the protons of Ser-77 in both the reduced and oxidized forms reveals that they are very similar to those experienced by Cys-77 in WT. On the basis of the hyperfine shifts observed for the Ser-77 protons and of the present structural analysis, it is concluded that the serine O gamma atom is coordinated to the polymetallic center, thus confirming the strict analogy of the electronic structures of the polymetallic center in both proteins. Capillary electrophoresis experiments demonstrate coordination of Ser-77 as an anion. Serine versus cysteine coordination in iron-sulfur proteins is briefly discussed.