NMR Analysis Shows That a b-Type Variant of Hydrogenobacter thermophilus Cytochrome c552 Retains Its Native Structure

Abstract

Conversion of

Photo-CIDNP NMR spectroscopy of a heme-containing protein

“…This information can be used as a guide in assigning the CIDNP spectrum, especially for residues far removed from the heme centre in the case of the holo proteins. The assignment of the observed resonances can be made by reference to the static accessibility data and by comparison with chemical shift assignments obtained from three-dimensional (3D) 15 N-edited TOCSY-HSQC and NOESY-HSQC experiments on 15 N-labelled reduced state holo C10A/C13A protein [17] and the wild-type c 552 [13]. These 3D NMR experiments have enabled the backbone amide protons and the indole NH protons of the tryptophan residues to be assigned.…”

“…The apo state of the protein is characterised by a decrease in helicity of 29% and an increase of 8.5% in the hydrodynamic radius, with respect to the holo form of the C10A/C13A mutant [16]. Both of these points and evidence from NMR spectroscopy [16,17] show that the apo state of the mutant can be characterised as a partially folded species with some molten-globule like characteristics which undergoes conformational fluctuations on a millisecond timescale. These observations are in close agreement with a similar investigation of the differences between the apo and holo forms of both cytochrome c [19] and myoglobin [20].…”

“…Previous studies have shown that the holo b-type protein retains a native-like fold, but is reduced in stability relative to the c-type protein. This is indicated by a decrease of 40 K in the thermal denaturation temperature and a lowering of the unfolding midpoint in denaturant titration experiments [15][16][17]. It has also been shown that the holo C10A/C13A mutant slowly and spontaneously loses the heme group, and subsequently undergoes amyloid fibril formation over a period of several weeks [18].…”

“…This in is contrast to cytochrome c in which the addition of heme to the apo protein only results in partial restoration of the native structure [19]. An NMR-based comparison of the holo C10A/C13A mutant and the wild-type c 552 protein shows that there are only very minor differences in both helical secondary structure and amide protection, indicating that the holo mutant and wild-type proteins have similar tertiary folds [17].…”

In Vitro Studies on Thioether Bond Formation between Hydrogenobacter thermophilus Apocytochrome c552 with Metalloprotoporphyrin Derivatives

“…In our previous work, we have reported the first in vitro formation of a c-type cytochrome containing two thioether bonds (4). It was shown that these bonds can form stereoselectively in vitro (5), presumably by forming a b-type cytochrome intermediate with the heme in one stereoisomeric orientation relative to its ␣,␥-mesoaxis (6). Some aspects of the heme orientation during the in vitro formation of c-type cytochromes have been discussed previously (7).…”