Transient binding of photodissociated carbon monoxide to CuB+ of eukaryotic cytochrome oxidase at ambient temperature. Direct evidence from time-resolved infrared spectroscopy

Biological electron transfer (eT) between redox-active cofactors is thought to occur by quantum-mechanical tunneling. However, in many cases the observed rate is limited by other reactions coupled to eT, such as proton transfer, conformational changes, or catalytic chemistry at an active site. A prominent example of this phenomenon is the eT between the heme groups of mitochondrial cytochrome c oxidase, which has been reported to take place in several different time domains. The question of whether pure eT tunneling in the nanosecond regime between the heme groups can be observed has been the subject of some experimental controversy. Here, we report direct observations of eT between the heme groups of the quinol oxidase cytochrome bo 3 from Escherichia coli, where the reaction is initiated by photolysis of carbon monoxide from heme o 3. eT from CO-dissociated ferrous heme o3 to the low-spin ferric heme b takes place at a rate of (1.2 ns) ؊1 at 20°C as determined by optical spectroscopy. These results establish hemeheme electron tunneling in the bo 3 enzyme, a bacterial relative to the mitochondrial cytochrome c oxidase. The properties of eT between the closely lying heme groups in the heme-copper oxidases are discussed in terms of the reorganization energy for the process, and two methods for assessing the rate of electron tunneling are presented.biological electron transfer ͉ heme-copper oxidases ͉ Marcus theory ͉ Moser-Dutton ruler ͉ ultrafast spectroscopy

Section: Resultsmentioning

confidence: 96%

Nanosecond electron tunneling between the hemes in cytochrome bo ₃

Jasaitis

Johansson

Wikström

et al. 2007

“…In a parallel kinetics study (6 By using TRIR, we have determined kL1 to be 4.7 x 105 so1 (1). These results reveal that CuB, in addition to its established electron transfer role, functions as a "ligand shuttle" in transporting CO (and plausibly other exogenous ligands including 02) to and from the heme of cyt a3.…”

mentioning

confidence: 79%

“…In a recent time-resolved infrared (TRIR) study (1) of the events after photodissociation of CO from cytochrome (cyt) a3 of reduced beef heart cytochrome oxidase (CcO), we reported conclusive evidence that photodissociated CO binds quantitatively to CuB at room temperature prior to equilibrating with solution. In a parallel kinetics study (6 By using TRIR, we have determined kL1 to be 4.7 x 105 so1 (1).…”

mentioning

confidence: 93%

Nature and functional implications of the cytochrome a3 transients after photodissociation of CO-cytochrome oxidase.

Woodruff

Einarsdóttir

Dyer

et al. 1991

114

117

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (1l is) as the postphotodissociation Cu'-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CUB to Fe,3 when CO binds to CUB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The timeresolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CUB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fe,3 pair. We speculate as to the identity ofthe ligand that is transferred between CUB and Fe,13 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.In a recent time-resolved infrared (TRIR) study (1) of the events after photodissociation of CO from cytochrome (cyt) a3 of reduced beef heart cytochrome oxidase (CcO), we reported conclusive evidence that photodissociated CO binds quantitatively to CuB at room temperature prior to equilibrating with solution. In a parallel kinetics study (6.E., P. M.

“…This reaction has usually been studied by the flow-flash technique (1, 2), which involves photodissociation of CO from the reduced cytochrome a3 in the presence of 02 (1,2). Time-resolved infrared experiments have shown that CO binds to CUB after photolysis of CO from cytochrome a3 (33,34). The pathway of CO into the reduced enzyme also involves the initial binding of CO to CUB (3,35), and recent studies indicate that the route for 02 may be similar (36)(37)(38).…”

Section: Methodsmentioning

confidence: 99%

Photodissociation of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)-cobalt(III)] complex: a tool to study fast biological reactions involving O2.

MacArthur

Sucheta²,

Chong³

et al. 1995

Upon photolysis at 355 nm, dioxygen is released from a (-peroxo)(,-hydroxo)bis[bis(bipyridyl)cobalt- (III)] complex in aqueous solutions and at physiological pH with a quantum yield of 0.04. The [Co(bpy)2(H20)z2+ (bpy = bipyridyl) photoproduct was generated on a nanosecond or faster time scale as determined by time-resolved optical absorption spectroscopy. A linear correspondence between the spectral changes and the oxygen production indicates that 02 is released on the same time scale. Oxyhemoglobin was formed from deoxyhemoglobin upon photodissociation of the (,iperoxo)(,u-hydroxo)bis[bis(bipyridyl)cobalt(III)I complex, verifying that dioxygen is a primary photoproduct. This complex and other related compounds provide a method to study fast biological reactions involving 02, such as the reduction of dioxygen to water by cytochrome oxidase.Many biological reactions involving dioxygen are so fast that studies by conventional stopped-flow techniques are impractical. Initiation of these and other fast reactions by photolysis offers a different approach. For example, the reaction of cytochrome c oxidase with dioxygen can be studied by a flow-flash method in which the reaction is initiated upon photodissociation of carbon monoxide (CO) bound to cytochrome a3 (1, 2). However, this method is potentially compromised by the fate of the photodissociated CO and, therefore, may not represent the physiological reaction of the enzyme under turnover conditions (3, 4). It is also not applicable to nonheme oxygen-activating systems. An alternative involves 02 production in situ by photodissociating synthetic caged dioxygen carriers. An ideal system would involve a direct source of dioxygen that does not react prematurely with the protein of interest but would release oxygen upon photodissociation on any relevant time scale. There should be no electrochemical or chemical reactions of the dioxygen carrier and its photoproduct with the protein, and interference from the photoproduct in the spectral region of the protein should be negligible. High solubility in aqueous solutions at physiological pH and high oxygen quantum yield are also desirable.Synthetic caged dioxygen carriers such as dicobalt ,u-peroxoor ,u-superoxo-bridged derivatives have the potential to fulfill the above requirements. Many of these complexes are soluble in aqueous solutions and are amenable for study by various spectroscopic probes (5-7). The most extensively studied have been cobalt(II) complexes of polyamines (8-13), amino acids (8, 14-16), and dipeptide ligands (8, 15, (11,15,18). The hydroxo bridge has been proposed to "lock in" the peroxo bridge, thereby shifting the equilibrium toward the oxygenated species (18). An advantage of the ,u-peroxo and ,-superoxo cobalt complexes is that such complexes have their major absorbances in the UV region. Therefore, electronic transitions of the protein in the visible region can be investigated without serious interference from transitions due to the cobalt complexes.Several studies of the photochemical...