Cytochrome c6 from Monoraphidium braunii. A cytochrome with an unusual heme axial coordination.
A soluble monoheme c-type cytochrome (cytochrome c6) has been isolated from the green alga Monoraphidium braunii. It has a molecular mass of 9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358 mV at pH 7. The determined amino acid sequence allows its classification as a class-I c-type cytochrome. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using 1H-NMR, ultraviolet/visible, EPR and Mössbauer spectroscopies. The pH equilibria are complex, several pKa values and pH-dependent forms being observed. The amino acid sequence, the reduction-potential value and the visible and NMR spectroscopies data in the pH range 4-9 indicate that the heme iron has a methionine-histidine axial coordination. However, the EPR and Mössbauer data obtained for the ferricytochrome show that in this pH range two distinct forms are present: form I, gz = 3.27, gy = 2.05 and gx = 1.05; form II, gz = 2.95, gy = 2.29 and gx = 1.43. While form I has crystal-field parameters typical of a methionine-histidine coordination, those associated with form II would suggest a histidine-histidine axial ligation. This possibility was extensively analyzed by spectroscopic methods and by chemical modification of a histidine residue. It was concluded that form II actually corresponds to an unusual type of methionine-histidine axial coordination. Straightforward examples of this type of coordination have recently been found in other c-type hemeproteins [Teixeira, M., Campos, A. P., Aguiar, A. P., Costa, H. S., Santos, H., Turner, D. L. & Xavier, A. V. (1993) FEBS Lett. 317, 233-236], corroborating our proposal. Since both forms, with very distinct crystal-field parameters, are shown to have the same reduction potential, it may be concluded that the axial and rhombic distortions of the heme-iron ligand field cannot be directly correlated with the heme-reduction potential. The pH-dependence studies have also shown that the form I and form II are interconvertible, with pKa approximately 5. To establish a possible physiological significance for this process, in particular for the interaction of the cytochrome with the membrane-bound electron-transfer complexes b6f and photosystem I, the effect of surfactants on the spectroscopic characteristics of cytochrome c6 has been studied.
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
A P
AP
Campos
A P
AP
Aguiar
M
M
Hervás
M
M
Regalla
J A
JA
Navarro
J M
JM
Ortega
A V
AV
Xavier
M A
MA
De La Rosa
M
M
Teixeira
D000595:Amino Acid Sequence; D000460:Chlorophyta / Q000737:chemistry*; D003580:Cytochromes / Q000737:chemistry*; D045348:Cytochromes f; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D013501:Surface-Active Agents / Q000737:chemistry
D003580:Cytochromes; D013501:Surface-Active Agents; D006418:Heme; D006639:Histidine; D045348:Cytochromes f; D008715:Methionine