Cardiovascular disease is the leading cause of death worldwide. Nitric oxide (NO) plays a fundamental role in cardiovascular health and disruptions in normal NO physiology is associated with the progression of cardiovascular disease. A relatively new mechanism by which heme proteins can support vasodilatation during hypoxia is by converting nitrite (NO2‐) to NO. This NO2‐ reductase activity plays essential roles in a variety of physiological processes. Because there is a large range in NO2‐ affinity and NO2‐ reductase activity in heme proteins with the same active site, this research seeks to better understand how the protein environment controls the binding chemistry of NO2‐ to the heme active site. Our goal is to determine how the distal pocket environment affects the binding affinity of NO2‐ to metmyoglobin mutants. Our hypothesis is that the distal pocket environment can increase the binding affinity of NO2‐ by way of electrostatic and steric interactions. To do this, we will compare the binding affinity of a series of distal pocket mutants that affect hydrogen bonding, polarity, and the size of the distal pocket. We will correlate coordination chemistry and binding affinity in order to establish a clear picture of how the protein environment controls NO2‐ binding affinity in heme proteins. We found that polar distal residues exhibit greater binding affinity to NO2‐ than non‐polar residues due to hydrogen bonding. These results indicate that proteins with polar distal residues will exhibit greater NO2‐ reductase activity. Understanding how the protein environment influences NO2‐ binding in heme proteins helps toward understanding how these proteins generate NO physiologically, and in designing therapeutics based on the NO2‐ reductase activity of heme proteins.