FULL PAPER The Conformational Flexibility of Oxidized Cytochrome c Studied through Its Interaction with NH 3 and at High Temperatures Lucia Banci, Ivano Bertini*, Georgios A. Spyroulias, and Paola Turano Department of Chemistry, University of Florence, Via Gino Capponi, 7, I-50121 Florence, Italy Fax: (internat.) + 39(0)55/275-7555 E-mail: bertini@lrm.fi.cnr.it Received November 21, 1997 Keywords: Cytochrome c / Hyperfine shift / Magnetic susceptibility anisotropy / NMR spectroscopy / Heme proteins The binding of ammonia to oxidized horse heart cytochrome c has been studied by 1 H-NMR, EPR, and CD spectroscopy at pH = 8.0. The affinity constant of the ligand is in the range 1.5-4 M -1 . The 1 H-NMR spectra of the heme group have been found to be similar to those of the high-pH forms, high- temperature forms, and cyanide adduct of the Met80Ala mu- tant of S. cerevisiae iso-1-cytochrome c. The assignment of a number of signals has led to the determination of the values The structure of the native cytochromes c has been exten- sively characterized by both X-ray crystallogra- phy [1] [2] [3] [4] [5] [6] [7] and NMR spectroscopy [8] [9] [10] [11] [12] . Several partially unfolded states of the oxidized species have also been detected spectroscopically as a function of tem- perature, pH, and denaturating agents [13-26] but the struc- tural characterization for these protein forms is not avail- able up to now. One important feature of cytochrome c folding/unfolding processes concerns the involvement of the covalently attached heme group and its axial ligands. The coordination of His and Met stabilizes the native state. In- deed, one of the first steps of ferricytochrome c unfolding upon increasing pH and/or temperature involves the de- tachment of Met80 from iron coordination [13-26] . In this respect NMR spectroscopy turns out to be a powerful tech- nique for the detection of changes in the iron coordination in ferricytochrome c because the hyperfine shifted reso- nances are heavily affected by changes in the axial coordi- nation of heme iron [27] [28] [29] [30] [31] [32] [33] . Indeed, through NMR spectroscopy, Hong and Dixon detected two forms of the alkaline isomerized species in slow exchange with the native form on the NMR time scale [34] [35] . At neutral pH and temperature above 42 °C, A ˚ ngstrom et al. [36] observed new species that closely resemble the alkaline isomerized forms. All these species contain low-spin iron(III). Recently, Taler et al. have further characterized the high-temperature species of horse cytochrome c and identified the 1 H-NMR resonances of the ε-CH 3 of Met80 in the high-temperature species at typical diamagnetic shift values [14] . It has been proposed that in oxidized cytochrome c at high pH values or at high temperatures the Nζ of a Lys residue substitutes Met80 in binding iron with a pK a of Eur. J. Inorg. Chem. 1998, 583-591  WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998 1434-1948/98/0505-0583 $ 17.50+.50/0 583 of the magnetic anisotropy and of the orientation of its axes. The latter are similar to those of the Met80Ala cyanide deri- vative. The assignment of the high-temperature species has been further pursued during this research. The analysis of the NMR data of the NH 3 adduct leads to the conclusion that substitution of Met80 at high pH or high temperature occurs through a ligand with cylindrical symmetry. This supports the suggestion that Met80 is substituted by a lysine at high pH. 8.5-9.5 [17] [34] [35] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . It is known that pyridine and imidazole bind to iron by replac- ing Met80 [50] [51] . We report here an investigation on NH 3 binding to horse heart cytochrome. The results are com- pared with those related to the high-temperature species, whose characterization has been further extended during this research, with those on the cyanide adduct of the Met80Ala mutant of S. cerevisiae cytochrome c, and with those on the alkaline forms. Results NH 3 Adduct The formation of the NH 3 adduct of horse heart cytochrome c (hh cyt c, hereafter) upon addition of increas- ing amounts of a solution of NH 4 Br was monitored by 1 H- NMR spectroscopy at pH = 8.0 and at 293 K. At this pH this protein is largely in its physiologically relevant confor- mation, as shown by its 1 H-NMR spectrum (Figure 1, A). Small traces of the alkaline forms are revealed by the peaks marked with asterisks. Upon addition of NH 4 Br new resonances appear at δ = 25.2, 23.5, 15.7, 15.5, 11.6, 10.9 whereas the signals of native hh cyt c decrease in intensity (Figure 1, B-D). A broad feature is observed upfield at δ =-11.9. All the above-mentioned resonances are due to non-exchangeable protons because they remain when the spectra are recorded in D 2 O. Analogous titration was performed by circular dichroism spectroscopy (CD) in the Soret region. In the native form, the CD spectrum (Figure 2, A) is characterized by a nega- tive absorption at 417 nm and a positive band at 400 nm, as already reported [40] .