Microsecond generation of oxygen-bound cytochrome c oxidase by rapid solution mixing

Area of Science:

• Biochemistry
• Bioenergetics
• Enzyme kinetics

Background:

• Current studies of cytochrome c oxidase (CcO) rely on carbon monoxide (CO) photolysis, a nonphysiological method.
• Evidence suggests CO may introduce artifacts into the enzyme's catalytic mechanism.

Purpose of the Study:

• To investigate the CcO catalytic reaction mechanism by directly mixing the enzyme with oxygen (O2) without using CO.
• To assess the influence of CO on early reaction intermediates and rates.

Main Methods:

• Development of a rapid solution mixer with a 20-microsecond mixing time.
• Utilizing resonance Raman scattering to detect spectral changes within 120 microseconds post-mixing.

Main Results:

• Direct mixing of CcO with O2 showed that CO does not affect the primary oxy-intermediate structure or its decay rate.
• CO also did not influence the oxidation rate of cytochrome a.
• These findings suggest CO impacts later stages of the catalytic process, potentially proton translocation.

Conclusions:

• The use of CO in previous studies may have led to artifacts in understanding the CcO catalytic mechanism.
• Direct mixing methods are crucial for re-examining CcO's reaction pathway.
• The developed rapid mixing technique is effective for studying biological reactions in the microsecond timescale.