Characterization of the structure and redox behaviour of cytochrome c3 from Desulfovibrio baculatus by 1H-nuclear-magnetic-resonance spectroscopy

Abstract

Complete assignment of the aromatic and haem proton resonances in the cytochromes c3 isolated from Desulfovibrio baculatus strains (Norway 4, DSM 1741) and (DSM 1743) was achieved using one- and two-dimensional 1H n.m.r. Nuclear Overhauser enhancements observed between haem and aromatic resonances and between resonances due to different haems, together with the ring-current contributions to the chemical shifts of haem resonances, support the argument that the haem core architecture is conserved in the various cytochromes c3, and that the X-ray structure of the D. baculatus cytochrome c3 is erroneous. The relative orientation of the haems for both cytochromes was determined directly from n.m.r. data. The n.m.r. structures have a resolution of approximately 0.25 nm and are found to be in close agreement with the X-ray structure from D. vulgaris cytochrome c3. The proton assignments were used to relate the highest potential to a specific haem in the three-dimensional structure by monitoring the chemical-shift variation of several haem resonances throughout redox titrations followed by 1H n.m.r. The haem with highest redox potential is not the same as that in other cytochromes c3.

Structural and functional characterization of cytochrome c3 from D. desulfuricans ATCC 27774 by 1H‐NMR

“…In fact, for the cytochromes c3 from the other Desulfovibrionaceae, haem IV has the highest redox potential [4,20,21] and, although for Dsm. baculatum cytochrome c3 haem III is also the less negative [22] and haem II has the lowest redox potential, the haems with similar redox potentials are I and IV [28].…”

“…As a result, the reported difference in midpoint redox potential for the last two haems to oxidise (120 mV) was considerably overestimated. In addition, the use of EPR studies together with differences in degree of haem exposure to solvent derived from the X-ray data to determine the structural cross-assignment of the redox potentials has already been shown to be unreliable [22].…”

“…When the intermolecular electron exchange is slow on the NMR time scale and the intramolecular electron exchange is fast [3,4], a separate set of NMR resonances is observed for the haem protons in each stage of oxidation (numbered according to the number of oxidised haems) with shifts which depend on the relative populations of each oxidised haem. This property is extremely useful since it can not only be used to determine the relative microscopic redox potentials for the different haems [3], but also provides the means to extend the structure-specific assignments from the spectra of the reduced form by the observation of chemical exchange connectivities between resonances of the same haem nucleus in the different stages of oxidation of the protein [3,4,[20][21][22]. In multiredox-centre proteins, this is a crucial piece of information for understanding their mode of action since it links unequivocally specific structural features with the respective redox potentials.…”

NMR Studies of Cooperativity in the Tetrahaem Cytochrome c3 from Desulfovibrio vulgaris

“…The solution potential does determine the relative concentrations of the different stages, and hence would affect the chemical shifts if intermolecular electron transfer were fast, as is the case in cytochrome c7 from Desulfomicrobium baculatum [27]. However, intermolecular electron transfer is slow in Dvc?, and therefore the solution potential affects only the intensity of the NMR signals.…”

Use of Paramagnetic NMR Probes for Structural Analysis in Cytochrome c3 from Desulfovibrio Vulgaris

“…A series of two-dimensional NMR studies has extended the number of both haem and non-haem proton assignments in the tetrahaem cytochromes c1 in reduced (diamagnetic) and in oxidised (paramagnetic) forms and allowed the cross-assignment of the individual haem redox potentials to the known X-ray structures Turner et al, 1992;Coutinho et al, 1992Coutinho et al, , 1993Coutinho et al, , 1995Park et al, 1991aPark et al, , b, 1996Piprra-Pereira et al, 1993;Sola and Cowan, 1992;Louro et al, 1996b). In recent studies of ferricytochroine c3 from DvH, the "C resonances of the haem substituents were also identified .…”