Evidence for structural heterogeneities and a study of exchange coupling. Mössbauer studies of cytochrome c1aa3 from Thermus thermophilus.

Fm Rusnak,E Münck,Ci Nitsche,Bh Zimmermann,Ja Fee

doi:10.1016/s0021-9258(18)49258-7

Fm Rusnak, E Münck + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)49258-7

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Abstract

We have studied samples of oxidized (as isolated) cytochrome c1aa3 from Thermus thermophilus in the pH range 5.7 to 9.3 with Mössbauer spectroscopy. In this pH range, the spectra of cytochromes c1 and a are independent of pH, whereas the spectra of cytochrome a3 are not. Most importantly, spectra taken in applied fields up to 6.0 T reveal the presence of multiple ferric forms of cytochrome a3. At any given pH value, at least two high-spin ferric cytochrome a3 species can be distinguished; in addition, most samples contain a low-spin ferric cytochrome a3 species (less than 20% of cytochrome a3). The Mössbauer spectra show clearly that all forms of cytochrome a3 are spin coupled (to CuB). We have analyzed the high field (H greater than or equal to 1.5 tesla) spectra of a sample at pH 6.5 in the framework of a model that considers isotropic exchange-coupling, JS1.S2, between a high-spin ferric (S1 = 5/2) cytochrome a3 and cupric CuB (S2 = 1/2). In strong applied fields, the spectra can be fitted with any value for J greater than or equal to 0.5 cm-1. In the strong coupling case (J/D1 approximately greater than 3), a zero field splitting parameter D1 approximately 2.5 cm-1 is required for cytochrome a3; this value is distinctly smaller than those observed for high-spin ferric hemes (4-20 cm-1). A model assuming weak coupling magnitude of J approximately 1 cm-1, yields D1 approximately 8 cm-1 and a parameter set for cytochrome a3 quite similar to that reported for metmyoglobin. A J-value of approximately 1 cm-1 does not demand the presence of a ligand bridging between cytochrome a3 and CuB.

Highlights

We havestudiedsamples of oxidized shown striking similarities between the two enzymes
A J-value of -1 cm” does They have succeeded in correlating a slow kinetic phase of not demand the presence of a ligand bridging between cyanide binding with the intensityof the g12 EPR signal and cytochrome u3and CuB
Our studies show that of integer electronic spin, i.e. the binuclear Fe3+-Cu2+center

Summary

Evidence for Structural Heterogeneities anad Study of Exchange Coupling

Sample at pH 6.5 in the framework of a mod$ that oxidized enzyme identified at least three conformations: 1) considersisotropicexchange-coupling, JSl*S,, be- the resting form, which normally is EPR-silent but is contween a high-spin ferric (S1= %) cytochrome u3 and verted intoa high-spin ferric species upon NO binding, 2) the. The objective of the present prokaryote Thermus thermophilus[1].This enzyme has been work was to investigate thspein-coupling between cytochrome shown to reduce oxygen to donors andcouple electron water in thepresence of electron transport to proton translocation u3 and Cus by fields (H > 0.1 studying samples in strong applied magnetic T)3over a wide range of temperatures. Recentoptical [3], EPR [3], resonance Raman [4], magnetic circular dichroism,’ and Mossbauer ( 5 , 6) studies of the oxidized form of the bacterial enzyme have. Mossbauer experiments for variable temperatre and applied fields were carried out as previously described [12].All isomer shifts, 6, are referred to iron metal at 298 K

RESULTS

DISCUSSION

Findings

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