Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli

Abstract

Cytochrome bd is a tri-heme (b 558, b 595, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O2 with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b 595 in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b 558 (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe2+-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b 558 (τ∼0.2–3 ms), and finally migrates from heme d to heme b 595 (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions.

Evidence for Fast Electron Transfer between the High-Spin Haems in Cytochrome bd-I from Escherichia coli

“…Whether the site also contains the second haem, b 595 , is not known with certainty. A large body of spectroscopic data suggests that haem b 595 indeed can form with haem d a di-haem active site [ 8 , 10 , 28 – 39 ] and that cytochromes b 595 and b 558 are each oxidised as the ‘oxy’ form decays (650 nm) during the low-temperature reaction with oxygen [ 40 ]. However, according to other reports, haem b 595 has an alternative or additional function [ 2 , 41 – 43 ].…”

“…The nanosecond interhaem electron transfer was detected in both cytochrome c and quinol oxidases of the haem-copper superfamily [ 46 , 49 , 50 ]. Under similar conditions, the 16-μs component of the photolysis-induced electron backflow was resolved in cytochrome bd -I [ 39 ]. In this work, we provide, to the best of our knowledge for the first time, evidence for the existence of a faster electron backflow component, on a submicrosecond time range, induced by photodissociation of CO from ferrous haem d in one-electron-reduced state of cytochrome bd -I.…”

Photosystem II and terminal respiratory oxidases molecular machines operating in opposite directions

“…Thus, although heme b 595 and heme d do not compose a structural binuclear site, one can speak of a functional di-heme site. The latter idea received support from spectroscopic and electrometric studies on cytochromes bd from E. coli and A. vinelandii (78,(166)(167)(168)(169)(170)(171)(172)(173)(174)(175)(176)(177)(178)(179)(180). Of interest, unlike cytochrome bd-I from E. coli (177), a bd-type oxidase from G. thermodenitrificans does not show any substantial excitonic interaction between heme b 595 and heme d (87).…”

“…The 3-μs phase was reported to be ratelimited by CO dissociation from Cu B (299), whereas the 1.4-ns and 35-μs phases are rate-limited by the electron tunneling between heme a 3 and heme a and between heme a and Cu A , respectively (135). Recently, evidence for the existence of a fast electron backflow component, on a submicrosecond time range, induced by photodissociation of CO from the ferrous heme d in one-electron-reduced state of cytochrome bd-I was provided (166,180). The electron from heme d first moves to heme b 595 and then reaches heme b 558 with the rate constant of ~16 μs (180).…”

“…Recently, evidence for the existence of a fast electron backflow component, on a submicrosecond time range, induced by photodissociation of CO from the ferrous heme d in one-electron-reduced state of cytochrome bd-I was provided (166,180). The electron from heme d first moves to heme b 595 and then reaches heme b 558 with the rate constant of ~16 μs (180). Thus, the mechanism of nanosecond interheme electron transfer may be universal not only in the family of heme-copper oxidases but also in the bd-type oxidases.…”