Kinetic Barriers to the Folding of Horse Cytochrome c in the Reduced State

Abstract

To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both excitation energy transfer from the lone tryptophan to the ferrous heme and far-UV circular dichroism (CD) indicate no accumulation of structural intermediates to a detectable level. Values of thermodynamic parameters obtained by two-state analysis of fluorescence transitions are DeltaG(H2O) = 11.65(+/-1.13) kcal x mol(-1) and C(m) = 3.9(+/-0.1) M GdnHCl in the presence of CO, and DeltaG(H2O)=19.3(+/-0.5) kcal x mol(-1) and C(m) = 5.1(+/-0.1) M GdnHCl in the absence of CO, indicating destabilization of ferrocytochrome c by approximately 7.65 kcal x mol(-1) due to CO binding. The native states of ferrocytochrome c and carbonmonoxycytochrome c are nearly identical in terms of structure and conformation except for the Fe2+-M80 --> Fe2+-CO replacement. Folding and unfolding kinetics as a function of GdnHCl, studied by stopped-flow fluorescence, are significantly different for the two proteins. Both refold fast, but carbonmonoxycytochrome c refolds 2-fold faster (tau = 1092 micros at 10 degrees C) than ferrocytochrome c. Linear extrapolation of the folding rates to the ordinate of the chevron plot projects this value of tau to 407 micros. The unfolding rate of the former in water, estimated by extrapolation, is faster by more than 10 orders of magnitude. Significant differences are also observed in rate-denaturant gradients in the chevron. Formation and disruption of the Fe2+-M80 coordination contact clearly impose high-energy kinetic barriers to folding and unfolding of ferrocytochrome c. The unfolding barrier due to the Fe2+-M80 bond provides sufficient kinetic stability to the native state of ferrocytochrome c to perform its physiological function as an electron donor.

A model for the misfolded bis-His intermediate of cytochrome c: the 1?56 N-fragment

“…As expected, the RR spectrum of the low spin heme of the N-fragment, closely resembles that of the imidazole complex of ferrous cyt c in SDS solutions [18]. Moreover, the same 6cLS species is observed also at pH 5.8 upon addition of 10 mM GuHCl to ferrous cyt c [18], whereas at higher concentration a 5cHS heme is already observed at neutral pH [33]. Fig.…”

Diffusional limits to the speed of protein folding: fact or friction?

“…We had the opportunity to study such a late stage process in horse cytochrome c [35]. Roder and coworkers have extensively characterized the equilibrium and kinetic properties of the MCO state of cytochrome c, a highly collapsed denatured globule from which folding to the native state can be exceedingly rapid [36][37][38]. This state is formed by chemically denaturing horse ferrocytochrome c, adding carbon monoxide (which binds to the haem iron), and then diluting the denaturant.…”

Viscosity-dependent structural fluctuation of the M80-containing Ω-loop of horse ferrocytochrome c

“…Within the limit of the stopped-flow resolution ($3 ms deadtime), the UCO refolds extremely fast in refolding buffer [64]. The kinetics exemplified in Fig.…”

“…The NCO-state is prepared from chemically denatured cyt c by lowering the denaturant concentration in the presence of carbon monoxide, which binds tightly to the heme iron and prevents formation of the native M80-heme (sulfur-iron) link [57,58,64]. Briefly, cyt c is unfolded in the presence of $6 M GdnHCl.…”

“…1). The slowness of the reaction allows accurate determination of k diss (s = 23 min with no addition, of the a-band [64]. Fig.…”