Interaction in cytochrome c oxidase between cytochrome a3 ligated with nitric oxide and cytochrome a.

Intramolecular electron transfer and conformational changes in cytochrome c oxidase were studied at room temperature following the photodissociation of CO bound to mixed-valence enzyme (cytochrome a3(2+)-CO CuB+ cytochrome a3+ CuA2+) and fully reduced enzyme. Time-resolved optical absorption difference spectra were collected in the Soret region on time scales of nanoseconds to milliseconds using a gated optical spectrometric multichannel analyzer. A global exponential fitting procedure combined with a singular value decomposition method was used to analyze the transient difference spectra at various times following CO photolysis. The analysis shows that at least two processes, with apparent lifetimes of 1.4 microseconds and 11.1 ms, are present following the photodissociation of CO bound to the fully reduced enzyme. These are attributed to a conformational change and CO recombination at the cytochrome a3 site, respectively. Global analysis of the mixed-valence CO complex transient difference spectra showed the presence of five intermediates with apparent lifetimes of 1.0 microseconds, 5.2 microseconds, 83.7 microseconds, 10.5 ms, and 25.3 ms. The data on a microsecond time scale are consistent with a mechanism involving a conformational change at cytochrome a3, followed by electron transfer from cytochrome a3 to cytochrome a with subsequent electron transfer to CuA. One of the two processes on a millisecond time scale is attributed to CO recombination and the other to a structural rearrangement or heme-heme electron transfer. On the basis of this mechanism, the kinetics and the absorption spectra of the intermediates involved in the conformational and electron transfer dynamics of the mixed-valence enzyme were determined.

Section: Discussionmentioning

confidence: 91%

Time-Resolved Optical Absorption Studies of Intramolecular Electron Transfer in Cytochrome c Oxidase

Georgiadis

Jhon²,

Einarsdóttir³

1994

“…The existence of a low-spin ferric heme iron near the spin probe may lead to line broadening of a spin-label signal which at cryogenic temperatures could provide information on iron-nitroxide distances [after Mascarenhas et al (1983)]. Because of the low-spin ferric heme, the nitroxide spin (here on the spin-label) could experience an internal electron spinspin interaction field, called (1/2∆H ss , which will be quantized either along or against the applied magnetic field.…”

Section: Resultsmentioning

confidence: 99%

Kinetics and Motional Dynamics of Spin-Labeled Yeast Iso-1-cytochrome c: 1. Stopped-Flow Electron Paramagnetic Resonance as a Probe for Protein Folding/Unfolding of the C-Terminal Helix Spin-Labeled at Cysteine 102

Vaughn

Sienkiewicz

et al. 1997

The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68-74], gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds. The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins. The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochrome c at the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype. This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme [Mchaourab, H. S. Lietzow, M. A., Hideg, K., & Hubbell, W. L. (1996) Biochemistry 35, 7692-7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P., & Millhauser, G. L. (1991) Biochemistry 30, 5515-5523]. The C-terminal cytochrome c helix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding. Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein. We observed an approximately 50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M. The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5. The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0. At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV-Vis that indicated a slower folding where heme misligation may be involved. Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction. The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics. The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted. The motion...

“…Recently, the cytochrome -cytochrome a3 distance was estimated as 12-16 Á by Ohnishi et al (1982) and 15 Á by Mascarenhas et al (1983) by analysis of dipolar spin-relaxation and EPR spectral line-shape changes, respectively, in nitric oxide complexes. Given the results presented here, the maximum distance that could be spanned by the three metal centers would be ~30 Á for a linear array: CuA-a-<z3.…”

Section: Resultsmentioning

confidence: 99%

Distance between the electron paramagnetic resonance-visible copper and cytochrome a in bovine heart cytochrome oxidase

Goodman¹,

Leigh²

1985

Electron paramagnetic resonance (EPR) at 15 K was used to probe the magnetic interaction between the visible copper CuA2+ and ferric cytochrome a in the carbon monoxide compound of beef heart cytochrome oxidase. At pH 8.6, the midpoint potentials (Em's) for one-electron oxidation of CuA+ and cytochrome a2+ were found to be 195 and 235 mV, respectively. Because the Em of CuA is well below that of cytochrome a under these conditions, the microwave power saturation of CuA could be measured as a function of percentage cytochrome a oxidized. Although progressive power saturation data directly provide only the product of the spin-lattice and transverse relaxation rates delta [1/(T1T2)], Castner's theory for the saturation of inhomogeneously broadened lines [Castner, T.G., Jr. (1959) Phys. Rev. 115 (6), 1506-1515], along with our own theoretical formulation of the dipolar T2, enabled us to determine the change in T1 of CuA due to dipolar relaxation by cytochrome a. The orientation of the principal g values of CuA with respect to those of cytochrome a was evaluated in partially oriented membranous multilayers. When allowance was made for uncertainties in the relative CuA-cytochrome a configuration and in the dipolar axis-magnetic field orientation, a range for the spin-spin distance r was calculated on the basis of the dipolar T1 of the gx component of CuA. This distance range was further restricted by consideration of T1 for the nonunique orientations of CuA giving rise to the gy signal. Only those values of r are possible for which the calculated T1 ratio (gx/gy) is equal to the experimentally determined ratio.(ABSTRACT TRUNCATED AT 250 WORDS)