PMID- 8555214
OWN - NLM
STAT- MEDLINE
DCOM- 19960223
LR  - 20141120
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 35
IP  - 2
DP  - 1996 Jan 16
TI  - Redox dependent interactions of the metal sites in carbon monoxide-bound 
      cytochrome c oxidase monitored by infrared and UV/visible spectroelectrochemical 
      methods.
PG  - 444-52
AB  - Spectroelectrochemical titration studies involving the binding of the 
      infrared-active probe ligand carbon monoxide (CO) to the heme alpha 3/CuB site of 
      bovine heart cytochrome c oxidase (CcO) have been reexamined. The 
      spectroelectrochemical cell employed was constructed to monitor both the infrared 
      (IR) and visible/Soret spectra of the CcO-CO complex as a function of the overall 
      oxidation state of the enzyme. A number of commonly used electron transfer 
      mediators were employed to shuttle electrons between the redox active sites 
      within the enzyme and the electrode surface. The well-documented shift in the CO 
      infrared stretch band maximum from 1963.3 cm-1 (CcO fully reduced) to 1965.5 cm-1 
      (CcO partially oxidized) was carefully titrated electrochemically. Deconvolution 
      of the asymmetric CO stretches indicates the existence of two different states of 
      CO vibrators within the enzyme, presumably due to two conformers which are 
      present in a ratio of approximately 5:1. Upon incrementally stepping the 
      potential from the fully reduced state to the partially oxidized state, we found 
      it possible to follow the decrease in the intensity of the original pair of these 
      conformers and the concomitant increase of a resultant pair while maintaining 
      this 5:1 ratio between the conformers. By plotting the change in the deconvoluted 
      CO peak intensities vs the redox potential, as well as the absorbance changes in 
      the visible/Soret spectra vs the redox potential, we found not only that both fit 
      an n = 1 electron process but also that the spectral changes tracked each other 
      identically with experimental error. Furthermore, analysis of the second 
      derivative of the Soret spectra allowed for the qualitative monitoring of the 
      oxidation state of the Fe alpha site which again tracked identically to that of 
      the CO shift in the IR region. These results would seem to confirm earlier 
      suggestions that perturbing the oxidation state of Fe alpha causes a 
      conformational change in the enzyme which affects the binding site for CO, namely 
      heme alpha 3. As a consequence of the CO IR stretching frequencies changing by 
      only 2 cm-1 during this redox titration, with no accompanying changes in half 
      band width, we suggest that it is impossible that this small but significant 
      change seen in the CO stretching frequencies could be due to an oxidation state 
      change in CuB, given the known sensitivity of the CO stretching frequency to 
      perturbations and the close proximity of Cu(B) to the CO binding site at heme 
      alpha 3 (4.5 A). Therefore, it would appear that Cu(B) must remain reduced as 
      long as CO is bound to the heme alpha 3 site. This is consistent with earlier 
      proposals that Fe alpha 3 and Cu(B) are acting together as a two-electron donor 
      to dioxygen.
FAU - Dodson, E D
AU  - Dodson ED
AD  - Department of Chemistry, Colorado State University, Fort Collins 80523, USA.
FAU - Zhao, X J
AU  - Zhao XJ
FAU - Caughey, W S
AU  - Caughey WS
FAU - Elliott, C M
AU  - Elliott CM
LA  - eng
GR  - GM 30306-05A3/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Ligands)
RN  - 0 (Metals)
RN  - 0 (Molecular Probes)
RN  - 42VZT0U6YR (Heme)
RN  - 7U1EE4V452 (Carbon Monoxide)
RN  - EC 1.9.3.1 (Electron Transport Complex IV)
SB  - IM
MH  - Animals
MH  - Binding Sites
MH  - Carbon Monoxide/*chemistry
MH  - Cattle
MH  - Electrochemistry
MH  - Electron Transport
MH  - Electron Transport Complex IV/*chemistry/metabolism
MH  - Heme/chemistry
MH  - In Vitro Techniques
MH  - Ligands
MH  - Metals/*chemistry
MH  - Molecular Probes
MH  - Myocardium/enzymology
MH  - Oxidation-Reduction
MH  - Spectrophotometry
MH  - Spectrophotometry, Infrared
MH  - Spectrophotometry, Ultraviolet
EDAT- 1996/01/16 00:00
MHDA- 1996/01/16 00:01
CRDT- 1996/01/16 00:00
PHST- 1996/01/16 00:00 [pubmed]
PHST- 1996/01/16 00:01 [medline]
PHST- 1996/01/16 00:00 [entrez]
AID - bi951313n [pii]
AID - 10.1021/bi951313n [doi]
PST - ppublish
SO  - Biochemistry. 1996 Jan 16;35(2):444-52. doi: 10.1021/bi951313n.