Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4).
P. stutzeri cytochrome c(4) is a di-haem protein, composed of two globular domains each with His-Met coordinated haem, and a hydrogen bond network between the domains. The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and different spin states of the oxidised haem groups. We have studied unfolding of oxidised P. stutzeri cyt c(4) induced thermally and by chemical denaturants. Horse heart cyt c was a reference molecule. Isothermal unfolding induced by guanidinium chloride and acid was followed by Soret, alpha/beta, and 701-nm band absorption, and by far-UV circular dichroism spectroscopy. Multifarious patterns emerge, but the two domains clearly unfold sequentially. One phase, assigned to unfolding of the N-terminal domain, proceeds at guanidinium concentrations up to approximately 1.0 M. This is followed by two overlapping phases at higher concentrations. The intermediate state maintains Fe-Met coordination, assigned to the C-terminal domain. Interdomain interaction is reflected in decreasing values of the cooperativity parameters. Differential scanning calorimetry shows a single peak, but two peaks appear when guanidinium chloride up to 0.4 M is present. This reflects different chemical action in chemical and thermal unfolding. Acid-induced unfolding kinetics was addressed by pH jumps using diode array stopped-flow techniques. Three kinetic phases in the 701 nm Fe-Met marker band, and four phases in the Soret and alpha/beta bands, spanning 4-1000 ms could be distinguished on pH jumps from 7.5 to the range 2.5-3.5. In this range of time and pH cyt c appears to unfold in no more than two phases. Spectral properties of the kinetic intermediates could be identified. Sequential domain unfolding, formation of high-spin states, and an intermediate state with Fe-Met coordination to a single haem group are features of the unfolding kinetics.
Andersen
Niels H
NH
Department of Chemistry, Building 207, Technical University of Denmark, DK-2800 Lyngby, Denmark.
Nørgaard
Allan
A
Jensen
Thomas J
TJ
Ulstrup
Jens
J
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry
D003574:Cytochrome c Group; C034186:cytochrome C4; D019791:Guanidine