Cyanide binding and active site structure in heme-copper oxidases: Normal coordinate analysis of iron-cyanide vibrations of CN− complexes of cytochromesba3 andaa3

Abstract

The cyanide isotope‐sensitive low‐frequency vibrations of ferrous cyano complexes of cytochrome a3 are studied for cytochrome ba3 from Thermus thermophilus and cytochrome aa3 from bovine heart. Cyanide complexes of ba3 display three isotope sensitive frequencies at 512, 485, and 473 cm−1. The first is primarily an Fe—C stretching motion, whereas the lower wavenumber modes are bending motions. These iron‐cyanide vibrations are independent of the redox levels of the other metal centers in the protein. On the other hand, the fully reduced bovine derivative complexed with cyanide gives rise to a bending vibration at 503 cm−1 and a stretching vibration at 469 cm−1. That is, the ordering of the stretching and bending frequencies is reversed from that of the bacterial protein. These results are analyzed by normal coordinate calculations to obtain comparative models for the binuclear O2 reducing site of the two proteins. We find that the observed frequencies are consistent with a linear Fe—C—N group and larger Fe—C stretching force constant (2.558 mdyn/Å) for ba3 and a slightly bent Fe—C—N group (angle ∼ 170°) and a smaller Fe—C stretching force constant (2.335 mdyn/Å) for aa3. Thus, there are significant differences in the interaction of cyanide with ferrous a3 in the two proteins that are most likely caused by a weaker proximal histidine interaction and stronger peripheral heme electron withdrawing effects in ba3. Possible sources of these protein‐induced effects are discussed. Using the analysis developed here, comparison of the FeCN stretching and bending frequencies of the ferrous bovine a3‐CN complex to those obtained from the ferric a3‐CN complex suggests that upon conversion of the resting to the fully reduced protein, a conformational change occurs that constrains the ligand binding site. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 1–15, 1998

Observation of Ligand Transfer in ba3 Oxidase from Thermus thermophilus: Simultaneous FTIR Detection of Photolabile Heme a32+–CN and Transient CuB2+–CN Complexes

“…Four 14 N atoms, presumably donated by histidine residues of the protein, provide a strong equatorial ligand field about Cu B and a second CN – is coordinated through the carbon atom to Cu B in an axial position. Although the Fe–CN modes were detected by RR spectroscopy in all of the CN-adducts, the corresponding Cu B –CN modes were not observed. − …”

“…Structural information of the O 2 -binding binuclear center of ba 3 oxidase and in cytochrome c oxidase (C c O) superfamily have been determined from a great number of spectroscopic and computational studies of the CO–, CN – , and NO-bound adducts. − In general, CO binds to the ferrous (Fe 2+ ) form of heme a 3 , whereas CN – and NO bind to both oxidized (Fe 3+ ) and reduced (Fe 2+ ) forms of heme a 3 . Because of the unusual ligand-binding kinetic properties of its binuclear center, cytochrome ba 3 from T. thermophilus is unique among the heme-copper oxidases in being susceptible to a detailed analysis of its ligation dynamics in the heme a 3 -Cu B site. − Resonance Raman (RR), electron nuclear double resonance (ENDOR), and electron paramagnetic resonance (EPR) spectroscopies, in conjunction with the permutations of 13 C- and 15 N-labeled cyanide have indicated that the reaction of oxidized ba 3 with cyanide yields a heme a 3 (II)-CN·Cu B (II)–CN complex. − It has been demonstrated that cytochrome ba 3 reacts slowly with excess HCN at pH 7.4 to create a form of the enzyme in which Cu A , heme b , and Cu B remain oxidized, while heme a 3 is reduced by one electron, presumably with the formation of cyanogen . The results supported a model in which one CN – binds through the carbon atom to ferrous heme a 3 , supporting a low-spin ( S = 0) configuration on the Fe; bridging by this cyanide to the Cu B is weak or absent.…”

“…The vibrational modes of cyanide when bound to heme-copper oxidases in various oxidation states have been studied by FTIR and RR spectroscopies. − In the oxidized form of the enzyme cyanide binds to the binuclear center to form a Fe 3+ –CN–Cu B 2+ complex in all heme-copper oxidases studied with the exception of the reports on ba 3 oxidase. − ,,,, There are some differences and conflicting reports on the fully reduced-CN complexes. In the bovine enzyme, modes at 2058 and 2045 cm –1 have been reported; in the partially reduced enzyme the modes at 2131 and 2093 cm –1 have been attributed to redox changes of the heme Fe–Cu B center or to protonation differences. , Of note, is the report of the modes observed at high CN – concentrations at 2093 and 2037 cm –1 assigned to the binding of a second CN – to Cu B .…”

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

“…Another intriguing feature of this CN-bound CcO structure is the significant bending of the Fe–C–N angle (96°). This bent structure is in line with previous resonance Raman characterizations and might be caused by the weak π-accepting ability of cyanide, which makes it easier to adopt a bent geometry compared to the isoelectronic Fe-CO complexes. − …”

Coherent two-dimensional infrared spectroscopy: Quantitative analysis of protein secondary structure in solution

“…Amide-I vibrations involve primarily combinations of backbone carbonyl stretches. 26,27 The 'local' carbonyl stretches, corresponding to individual amino acids within the protein, form the basis for the delocalized normal modes. 28 The frequencies of the normal modes are given by the frequencies of the local C]O stretches as well as the magnitude of the couplings between these local oscillators.…”