Solution Structure and Dynamics of the Functional Domain of Paracoccus denitrificans Cytochrome c₅₅₂ in Both Redox States^,

Abstract

A soluble and fully functional 10.5 kDa fragment of the 18.2 kDa membrane-bound cytochrome c(552) from Paracoccus denitrificans has been heterologously expressed and (13)C/(15)N-labeled to study the structural features of this protein in both redox states. Well-resolved solution structures of both the reduced and oxidized states have been determined using high-resolution heteronuclear NMR. The overall protein topology consists of two long terminal helices and three shorter helices surrounding the heme moiety. No significant redox-induced structural differences have been observed. (15)N relaxation rates and heteronuclear NOE values were determined at 500 and 600 MHz. Several residues located around the heme moiety display increased backbone mobility in both oxidation states, while helices I, III, and V as well as the two concatenated beta-turns between Leu30 and Arg36 apparently form a less flexible domain within the protein structure. Major redox-state-dependent differences of the internal backbone mobility on the picosecond-nanosecond time scale were not evident. Hydrogen exchange experiments demonstrated that the slow-exchanging amide proton resonances mainly belong to the helices and beta-turns, corresponding to the regions with high order parameters in the dynamics data. Despite this correlation, the backbone amide protons of the oxidized cytochrome c(552) exchange considerably faster with the solvent compared to the reduced protein. Using both differential scanning calorimetry as well as temperature-dependent NMR spectroscopy, a significant difference in the thermostabilities of the two redox states has been observed, with transition temperatures of 349.9 K (76.8 degrees C) for reduced and 307.5 K (34.4 degrees C) for oxidized cytochrome c(552). These results suggest a clearly distinct backbone stability between the two oxidation states.

Correlation between the Stability and Redox Potential of Three Homologous Cytochromescfrom Two Thermophiles and One Mesophile

“…A similar situation occurs in the homologous HT c 552 and PH c 552 proteins. The increased stability upon one-electron reduction of the present proteins should be attributed to an as-yet undefined local environment near the heme, as observed in Paracoccus denitrificans cyt c. 18) More elaborate microcalorimetry and hydrogen exchange studies of the three cyts c should eventually lead to a dynamic and systematic explanation of the overall stability differences between the two redox forms.…”

The Electron Transfer Complex between Cytochrome c552 and the CuA Domain of the Thermus thermophilus ba3 Oxidase

“…S1 in the supplemental data) had shown a 20-nm shift of the tryptophan band (42). This is apparently due to an alteration in the electronic structure of Trp 57 , since the protein conformation is identical in both redox states as confirmed by both x-ray and NMR structure analysis (42,43), thus excluding an explanation that is based on conformational changes in the protein structure. Hence, the only distinction that could explain this redox state-dependent effect in the fluorescence spectrum of P. denitrificans cytochrome c 552 is the additional electron delocalized across the porphyrin system.…”

“…In the case of P. denitrificans, for example, fluorescence spectra of reduced and oxidized cytochrome c 552 (see Fig. S1 in the supplemental data) had shown a 20-nm shift of the tryptophan band (42). This is apparently due to an alteration in the electronic structure of Trp 57 , since the protein conformation is identical in both redox states as confirmed by both x-ray and NMR structure analysis (42,43), thus excluding an explanation that is based on conformational changes in the protein structure.…”

Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers

“…The three-dimensional structure of R. capsulatus cyt c y is not available, but its amino acid sequence is highly similar (63% identity and 75% similarity over 95 amino acids) to that of cyt c 552 of Paracocus denitrificans of known structure (32,33). A structural model for the cyt c domain of R. capsulatus cyt c y built by homology modeling based on that of cyt c 552 indicated that Lys-19 is on the surface of the protein and solvent exposed, whereas His-53 is slightly buried into the protein (Fig.…”