Tapan Kanti Das

Time-resolved study of tryptophan fluorescence in vesicle reconstituted cytochrome oxidase Effect of redox transition

Time‐resolved study of fluorescence decay of the tryptophan residue in bovine cytochrome c oxidase in phospholipid vesicles is reported for the first time. The effect of the redox state of the protein on its conformation has been investigated using time‐resolved decay of tryptophan fluorescence in the oxidised and reduced protein. The fluorescence decay was best fitted using a discrete three exponential model. Amplitude distribution of lifetimes also showed three distinct regions in the analysis of decay profiles by the maximum entropy method (MEM). Results of the time resolved studies showed that the amplitudes as well as the lifetimes of the trytophan fluorescence remain the same for the oxidised and the reduced states ofcytoehrome c oxidase, indicating that the environment around tryptophan residues remains more or less unaltered on reduction of the protein. The results suggest that there is no global conformational change in the protein on electron transfer and support the possibility of the existence of local fluctuations in the protein during the redox cycle. p]Cytochrome c oxidase; Tryptophan fluorescence; Conformational change

Conformational change due to reduction of cytochrome-c oxidase in lauryl maltoside: picosecond time-resolved tryptophan fluorescence studies on the native and heat modified enzyme

Detailed fluorescence studies on bovine heart cytochrome-c oxidase (CcO) has been carried out in lauryl maltoside solution. Steady-state fluorescence of the tryptophan residues of the enzyme showed that the fluorophores are embedded deep inside the hydrophobic protein cavity. Time resolved studies of tryptophan fluorescence of native and heat treated CcO have been carried out in both reduced and oxidised forms using synchronously pumped pulsed picosecond dye laser and single photon counting technique. Decay of the tryptophan fluorescence have been fitted using discrete four exponential model. Amplitude distribution of lifetimes also showed four distinct regions in the analysis of the decay profiles by maximum entropy method (MEM). The results indicate that controlled heat treatment of CcO affects the conformation of the enzyme near the active centers which makes it incapable of active proton pumping while the electron transfer property is still conserved. Reduction of the native CcO is associated with a large conformation change in lauryl maltoside near the active centers which is not observed in case of CcO encapsulated in vesicles. Reduction of the heat treated enzyme was found to have a conformation different from the reduced native CcO.

Micelle-induced release of haem–NO from nitric oxide complex of myoglobin

The cleavage of the haem-proximal histidine bond and concomitant release of haem–NO from MbNO has been achieved at physiological pH by aqueous micelles such as hexadecyltrimethyl ammonium bromide and sodium dodecyl sulfate, and the released haem–NO complex is encapsulated in the hydrophobic micellar cavity; this micellar effect has close similarity with the action of guanyl cyclase on nitrosyl haem proteins.

Redox‐linked conformational changes in bovine heart cytochrome c oxidase: Picosecond time‐resolved fluorescence studies of cyanide complex

Picosecond time-resolved fluorescence studies are carried out on cyanide-inhibited and heat-modified cytochrome c oxidase in aqueous lauryl maltoside surfactant solution, as well as in an aqueous vesicle, to understand the conformational changes associated with electron transfer and proton pumping activity of the enzyme. The tryptophan fluorescence decay profiles follow a four exponential model, which also matches the lifetime maxima obtained in a maximum entropy method analysis. The fast lifetime components are highly affected by the reduction and chemical modification of the enzyme. Changes in these lifetime components are related to the conformational changes in the vicinity of the heme centers of the enzyme. The cyanide-inhibited enzyme in the oxidized form shows a fluorescence decay profile similar to that of the native oxidized form, indicating that the conformational changes due to cyanide binding are very small. However, reduction of the cyanide-inhibited enzyme that leaves cyanide bound heme alpha3 oxidized causes a large increase in the fluorescence lifetimes, which indicates very significant conformational changes due to electron transfer to the dinuclear Cu(A) and heme alpha centers. A comparison of the tryptophan fluorescence decay of various other modified forms of the enzyme leads us to propose that the possible site of conformational coupling is located near heme alpha instead of the binuclear heme alpha3-Cu(B) center.

Heme CD as a probe for monitoring local structural changes in hemeproteins: alkaline transition in hemeproteins

Structural change due to acid-alkaline transition in hemeproteins were monitored by circular dichroism measurements in the Soret region. It was observed that in cytochrome c and horseradish peroxidase, alkaline transition results in a large change in the heme CD due to significant conformational change in the heme cavity region. In metmyoglobin a simple protolytic mechanism associated with alkaline transition involves very small conformational changes.

Unfolding pathway of cytochromec oxidase induced by ionic surfactants: Circular dichroism and picosecond time-resolved fluorescence studies

The unfolding of the membrane protein, cytochromec oxidase (CcO) induced by ionic surfactants have been studied by using circular dichroism, optical absorbance and time resolved tryptophan fluorescence spectroscopic methods. Ionic surfactant cetyltrimethyl ammonium bromide (CTAB) was found to cause denaturation of this membrane protein leading to release of both, the hemea residues from CcO indicated by both CD and optical titration. Upon dissociation of the hemes from the protein matrix; the tryptophan fluorescence intensity of CcO increased drastically and the fluorescence lifetimes became much longer compared to the short lifetimes observed in the native protein. The shortest lifetime of 70 ps observed in the native protein due to strong quenching (energy transfer) of the heme groups, increased ∼10-fold in the CTAB-unfolded protein indicating complete removal of the heme groups from the protein matrix. Remarkable differences were observed between the mode of actions of ionic surfactants and the commonly used denaturant guanidine hydrochloride. Improved data analysis of maximum entropy method showed that the lifetime distribution pattern in the two cases of unfolding were very different. The lifetimes in guanidine hydrochloride unfolded CcO were much shorter and more widely distributed indicating that the hemes are probably not separated away from the protein matrix and that the unfolded state is highly heterogeneous. Our results further showed that the lauryl maltoside inhibits denaturation of CcO by the ionic surfactant and the initial step of the denaturation possibly involves quantitative replacement of the lauryl maltoside by the ionic surfactant at the surface of the enzyme.

Erratum to ‘conformational change due to reduction of cytochrome-c oxidase in lauryl maltoside: picosecond time-resolved tryptophan fluorescence studies on the native and heat modified enzyme’ [Biochim. Biophys. Acta 1209 (1994) 227–237]

Section 1. 228 1 15 228 1 25 228 1 26 228 1 33 228 1 35 228 1 35 228 1 36 228 1 42 Section 2.2. 228 2 2 Section 2.3. 228 2 5 Section 2.4. 228 2 2 229 1 3 229 1 28 Section 3. 230 2 22 230 2 27 231 1 1 Section 3.1. 231 2 30 231 2 35 Section 3.2. 232 2 15 233 2 13 Section 4.1. 235 1 7 235 2 29 Section 4.2. 236 1 9 Section 4.3. 236 1 4 ...has been shown [13,14]... ...change in the enzyme [ 15,16]... ...It has been shown [15]... ...13 subunits [17]... ...such as myoglobin [ 18,19] .... ...hemoglobin [20,21] .... ...bacteriorhodopsin [22]... ...tryptophan residues [23]...

Time-resolved fluorescence study of the single tryptophan in thiocyanate and azide derivatives of horseradish peroxidase: Implication for apH-induced conformational change in the heme cavity

Detailed pH-dependent steady state and picosecond time-resolved tryptophan fluorescence studies on thiocyanate and azide complexes of horseradish peroxidase have been carried out. The fluorescence decay of the single tryptophan in these species was fitted to a discrete three exponential model. Maximum entropy method analysis also gave three distinct regions of lifetime distributions. The fast subnanosecond lifetime component was found to have > 97% amplitude contribution while other two longer lifetime components have small contributions. Small contributions from the nanosecond lifetime components possibly arise from apoprotein impurity or some small amount of disordered heme conformer of the protein. pH dependence of the fast picosecond lifetime components was found to show a systematic behavior which has been interpreted in the light of obligatory conformation change associated with activation of the enzyme at low pH.