Abstract
The proton NMR spectra of ferricytochrome c' from Rhodopseudomonas palustris, Rhodospirillum molischianum, Rhodospirillum rubrum, and Chromatium vinosum have been investigated for the purpose of further elucidating the common spectral and/or structural properties for this subclass of cytochromes in the acidic and alkaline forms, and to characterize in detail the dynamics and structural basis for this acid in equilibrium with alkaline transition. The identification of strongly upfield-shifted meso-H peaks in all but C. vinosum ferricytochrome c' at weakly acidic to neutral pH is consistent with, but not proof for, S = 3/2 character for the spin state of C. vinosum, but argues for primarily S = 5/2 character for the other three proteins. Hence, we conclude that the quantum mechanically mixed S = 3/2, S = 5/2 spin ground state of neutral pH C. vinosum ferricytochrome c' is an anomaly rather than a characteristic of this class of proteins. The 1H NMR spectra of ferricytochromes c' at alkaline pH again exhibit strong similarities among all members except that for C. vinosum. Two pK values are observed for ferricytochrome c' for R. molischianum and C. vinosum, of which the higher value pK is accompanied by significant line broadening, as found earlier for the proteins from both R. rubrum and R. palustris. Detailed analysis of the exchange line broadening for all four proteins reveals that hydrolysis is the rate-limiting step, with base catalysis occurring at about the same rate in the diffusion control limit for all four proteins. The variable first order dissociation rates of the alkaline species reveal differential stabilities of that species in the order R. palustris greater than R. molischianum greater than R. rubrum much greater than C. vinosum. The rates of exchange of the axial His imidazole labile proton was determined by linewidth and saturation transfer analysis and shown to occur via base catalysis at the same diffusion control rate as found for the acid----alkaline transition for the oxidized protein, and support the proposal that the acid----alkaline transition involves simply the abstraction of a proton from the neutral His imidazole to yield an imidazolate.
Highlights
The proton NMR spectra of ferricytochrome c’ from Rhodopseudomonas palustris, Rhodospirillum molischianum, Rhodospirillum rubrum, and Chromatium vinosum have been investigated for the purpose of further elucidating the common spectral and/or structural properties for this subclass of cytochromes in the acidic and alkaline forms, and to characterize in detail the dynamics and structural basis for this acid e alkaline transition
One implied characteristic property of ferricytochromes c’ is a neutral pH spin ground state which is a quantum mechanical mixture of S = 3/2 and S = 5/2 states that results in a reduced magnetic moment and an unusual ESR spectrum [10, 15,16,17,18,19]
Location of Meso-H Resonances-The complete ‘H NMR spectra of R. palustris and R. rubrum ferricytochromes c’ at low pH are shown in Fig. 2, A and D, respectively
Summary
The proton NMR spectra of ferricytochrome c’ from Rhodopseudomonas palustris, Rhodospirillum molischianum, Rhodospirillum rubrum, and Chromatium vinosum have been investigated for the purpose of further elucidating the common spectral and/or structural properties for this subclass of cytochromes in the acidic and alkaline forms, and to characterize in detail the dynamics and structural basis for this acid e alkaline transition. The majority of cytochromes c’ are dimeric, with extensive spectroscopic studies reported on the species from Rhodospirillum rubrum, Chromatium vinosum, and Rhodospirillum moldschiunum [9,10,11,12,13,14,15,16,17,18,19,20,21,22,23] The latter protein has yielded a high resolution x-ray crystal structure [24, 25] that confirms a fivecoordinated heme in a folded polypeptide quite different from cytochrome c, but remarkably similar to that of Escherichia coli cytochrome bs6*(26). The hyperfine shifts observed for primarily heme and axial histidyl protons have been shown to serve as sensitive indicators of a variety of molecular/
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