RUI: Novel Heme Chemistry of Cytochrome c'

Eastern Oregon University, La Grande OR

Investigators

Abstract

In many processes vital for life (e.g. respiration, gas sensing/signaling, and toxicity defense) heme proteins must bind a specific diatomic gas, e.g. oxygen (O2), carbon monoxide (CO), or nitric oxide (NO). To modulate heme reactivity (and achieve a selective gas response), proteins utilize the structural and chemical properties of the heme pocket environments. A major goal of this project is to investigate how the spatial environments of heme iron sites can dramatically affect their reactivity with gases. This knowledge is important for understanding fundamental biochemical reactions in animals, plants, and bacteria, as well as for the rational design of heme-gas sensors with unique properties. Undergraduate research is an integral part of the project, providing students and local high-school teachers with stimulating experiences and motivation for future scientific endeavors. The project will focus on the novel heme reactivity of bacterial cytochromes c' in which steric constraints significantly disfavor the distal coordination of O2, CO, and NO, while promoting an unusual distal to proximal heme-NO conversion. The coordination of NO at both heme faces (distal and proximal) represents a new type of heme protein reactivity that may be relevant to the proposed NO-binding function(s) of cytochromes c', as well as to NO-selective responses in gas-sensing proteins. By characterizing distal pocket variants of Alcaligenes xylosoxidans cytochrome c' (AXCP), as well as other cytochromes c', the overall goal is to define the connection between distal steric constraints, structural perturbations, and heme reactivity, including distal vs proximal NO coordination. A "loaded spring" hypothesis will be tested in which steric clashes between the crowded distal pocket and diatomic gas (XO) ligands lead to structurally perturbed high-energy heme-XO complexes with altered reactivity. The project will utilize spectroscopy (UV-visible absorption, resonance Raman, and Electron Paramagnetic Resonance), kinetics (stopped-flow and flash photolysis) and other techniques (X-ray crystallography and potentiometry) to study wild-type and variant cytochrome c' reactivity as function of distal steric bulk and gas identity. Undergraduates and high school teachers will receive training in these techniques and be involved in interpreting the results.