Cytochrome c' from Rhodospirillum rubrum has been studied by proton magnetic resonance (NMR) at 270 MHz. The pH and temperature-dependence properties as well as proton water relaxation enhancement and bulk susceptibility measurements were examined. We conclude that the fifth ligand to the iron is histidine. The pH-dependent shift of the heme methyl resonances of the ferric protein shows pKa's at 5.8 and 8.7. The low-pH equilibrium causes only minor changes in the properties of the protein. However, the high-pH equilibrium causes large changes throughout the NMR spectra which correlate with the reported visible spectral changes. These NMR spectral changes are compared with the low-temperature EPR and Mössbauer spectroscopic data. Analyses of the NMR data show that a second histidine, which is present in the sequence of c' from R. rubrum but is not conserved in other cytochromes c', is not a "distal" histidine. The nature of the sixth ligand and the significance of the high-pH transition are discussed.

Measurements of the proton relaxation rates of lanthanide( 111) aquo-ion solutions show that the correlation times, which govern the proton relaxation times in these solutions, are very short (ca. 1 0-13 s), and are predominantly due to the efectron-spin relaxation times of the cations. The variations in the relaxation rates for different Ln"I ions are discussed in terms of modulation of the different ligand-field splittings. The temperature dependence of the relaxation rates has also been studied and a relationship to the magnitude of ligand-field splittings of the different Ln"' ions is discussed.

The class I I cytochromes Rhodospirillum molischianum cytochrome c', Rho (/op.veud~)n.lona.r puluslris cytochrome c556 and Agrohacterium tumefuciens (B2a) cytochrome cSs(, have been investigated with a variety of spectroscopic techniques. The cytochrome c' was found to be high-spin and the two cytochromes ~5 5 6 were found to be mainly low-spin and sx-coordinate with the fifth and sixth ligands being histidine and methionine. The implications of the different types of iron coordination arc discussed.The cytochromes c comprise a group of haemproteins originally defined through possession of a characteristic absorption at about 550 nm in visible spectra of their reduced forms [I]. This spectral feature arises from covalently bound protoporphyrin IX. I n all cytochromes c so far investigated thioether bonds between the haem vinyl substituents and cysteine residues of the protein provide the covalent linkages. In all cases too, a histidine residue adjacent to one of the cysteine residues provides a ligand to the iron atom. The amino acid sequence of a typical haem-binding site in the c-type cytocliromes is -Cys-Xaa-Xaa-Cys-His-. The simple correlation of an absorption band at 550 nm and the covalent haem linkage only applies to low-spin cytocliromes c. A more precise characterisation requires isolation of the haem moiety [2,31. As knowledge concerning the cytochrome c group of proteins has increased (see 12-81 for good reviews) clear sub-divisions of this group have become apparent. A subclassification scheme has been proposed [9] based on the amino acid sequences of a large number of proteins.The most studied type of cytochrome c is the group of class I cytochromes c which includes mitochondria1 cytochrome c and which is defined by possession of a single haem group attached close to the N terminus of the protein with the sixth iron ligand known or presumed to be provided by a metliionine residue about three-quarters of the way along the sequence. These proteins are low-spin and have the 550-nm band. Class I1 cytochromes c on tlie other hand have their covalent haem attachment site near tlie C terminus. This group includes some low-spin proteins with n band at 550 nni and the predominantly high-spin proteins, the cytochromes c' (see below), which have very different absorption spectra.Comparatively little is known about class I1 cytochromes c relative to the class I proteins; although the amino acid sequences of 11 class I1 cytochromes c have been determined [8 -151 (and R. P. Ambler, unpublished work) and an X-ray structure for Rhodospirillum molischianum ferricytochrome c' is available [16]. Even the biochemical functions of class 11Ahhreviutions. EPR, electron paramagnetic resonance; NM R. nuclear magnetic resonance. cytochromes c have not been established although they have been suggested to operate in electron-transport chains, either as simple electron-transfer proteins or as terminal oxidases [1,2]. Our interest in the class I1 cytochromes c concerns the nature of their iron coordination centres.Only two of...

No abstract

No abstract